T1r3 a novel taste receptor

ABSTRACT

The present invention relates to the discovery, identification and characterization of a receptor protein, referred to herein as T1R3, which is expressed in taste receptor cells and associated with the perception of bitter and sweet taste. The invention encompasses T1R3 nucleotides, host cell expression systems, T1R3 proteins, fusion protein, transgenic animals that express a T1R3 transgene, and recombinant “knock-out” animals that do not express T1R3. The invention further relates to methods for identifying modulators of the T1R3-mediated taste response and the use of such modulators to either inhibit or promote the perception of bitterness or sweetness. The modulators of T1R3 activity may be used as flavor enhancers in foods, beverages and pharmaceuticals.

BACKGROUND

[0001] The present invention relates to the discovery, identificationand characterization of a G protein coupled receptor, referred to hereinas T1R3, which is expressed in taste receptor cells and associated withthe perception of sweet taste. The invention encompasses T1R3nucleotides, host cell expression systems, T1R3 proteins, fusionproteins, polypeptides and peptides, antibodies to the T1R3 protein,transgenic animals that express a T1R3 transgene, and recombinant“knock-out” animals that do not express T1R3. The invention furtherrelates to methods for identifying modulators of the T1R3-mediated tasteresponse and the use of such modulators to either inhibit or promote theperception of sweetness. The modulators of T1R3 activity may be used asflavor enhancers in foods, beverages and pharmaceuticals.

[0002] The sense of taste plays a critical role in the life andnutritional status of humans and other organisms. Human taste perceptionmay be categorized according to four well-known and widely accepteddescriptors, sweet, bitter, salty and sour (corresponding to particulartaste qualities or modalities), and two more controversial qualities:fat and amino acid taste. The ability to identify sweet-tastingfoodstuffs is particularly important as it provides vertebrates with ameans to seek out needed carbohydrates with high nutritive value. Theperception of bitter, on the other hand, is important for its protectivevalue, enabling humans to avoid a plethora of potentially deadly plantalkaloids and other environmental toxins such as ergotamine, atropineand strychnine. During the past few years a number of molecular studieshave identified components of bitter-responsive transduction cascades,such as α-gustducin (1, 2), Gγ13 (3) and the T2R/TRB receptors (4-6).However, the components of sweet taste transduction have not beenidentified so definitively (7, 8), and the elusive sweet-responsivereceptors have neither been cloned nor physically characterized.

[0003] Based on biochemical and electrophysiological studies of tastecells the following two models for sweet transduction have been proposedand are widely accepted (7, 8). First, a GPCR-G_(s)-cAMP pathway—sugarsare thought to bind to and activate one or more G protein coupledreceptors (GPCRs) linked to G_(s); receptor-activated Gα_(s) activatesadenylyl cyclase (AC) to generate cAMP; cAMP activates protein kinase Awhich phosphorylates a basolateral K⁺ channel, leading to closure of thechannel, depolarization of the taste cell, voltage-dependent Ca⁺⁺ influxand neurotransmitter release. Second, a GPCR-G_(q)/GβΓ-IP₃pathway—artificial sweeteners presumably bind to and activate one ormore GPCRs coupled to PLCβ2 by either the α subunit of G_(q) or by Gβγsubunits; activated Gα_(q) or released Gβγ activates PLCβ2 to generateinositol trisphosphate (IP₃) and diacyl glycerol (DAG); IP₃ and DAGelicit Ca⁺⁺ release from internal stores, leading to depolarization ofthe taste cell and neurotransmitter release. Progress in this field hasbeen limited by the inability to clone sweet-responsive receptors.

[0004] Genetic studies in mice have identified two loci, sac (determinesbehavioral and electrophysiological responsiveness to saccharin, sucroseand other sweeteners) and dpa (determines responsiveness toD-phenylalanine), that provide major contributions to differencesbetween sweet-sensitive and sweet-insensitive strains of mice (9-12).Sac has been mapped to the distal end of mouse chromosome 4, and dpamapped to the proximal portion of mouse chromosome 4 (13-16). The orphantaste receptor T1R1 was tentatively mapped to the distal region ofchromosome 4, hence, it was proposed as a candidate for sac (17).However, detailed analysis of the recombination frequency between T1R1and markers close to sac in F2 mice indicates that T1R1 is ratherdistant from sac (˜5 cM away according to genetic data of Li et al (16);and more than a million base pairs away from D18346, the marker closestto sac. Another orphan taste receptor, T1R2, also maps to mousechromosome 4, however, it is even further away from D18346/sac than isT1R1.

[0005] To thoroughly understand the molecular mechanisms underlyingtaste sensation, it is important to identify each molecular component inthe taste signal transduction pathways. The present invention relates tothe cloning of a G protein coupled receptor, T1R3, that is believed tobe involved in taste transduction and may be involved in the changes intaste cell responses associated with sweet taste perception.

SUMMARY OF THE INVENTION

[0006] The present invention relates to the discovery, identificationand characterization of a novel G protein coupled receptor referred tohereafter as T1R3, that participates in the taste signal transductionpathway. T1R3 is a receptor protein with a high degree of structuralsimilarity to the family 3 G protein coupled receptors (herein afterGPCR). As demonstrated by Northern: Blot analysis, expression of theT1R3 transcript is tightly regulated, with the highest level of geneexpression found in taste tissue. In situ hybridization indicates thatT1R3 is selectively expressed in taste receptor cells, but is absentfrom the surrounding lingual epithelium, muscle or connective tissue.Moreover, T1R3 is highly expressed in taste buds from fungiform, foliateand circumvallate papillae.

[0007] The present invention encompasses T1R3 nucleotides, host cellsexpressing such nucleotides and the expression products of suchnucleotides. The invention encompasses T1R3 protein, T1R3 fusionproteins, antibodies to the T1R3 receptor protein and transgenic animalsthat express a T1R3 transgene or recombinant knock-out animals that donot express the T1R3 protein.

[0008] Further, the present invention also relates to screening methodsthat utilize the T1R3 gene and/or T1R3 gene products as targets for theidentification of compounds which modulate, i.e., act as agonists orantagonists, of T1R3 activity and/or expression. Compounds whichstimulate taste responses similar to those of sweet tastants can be usedas additives to act as flavor enhancers in foods, beverages orpharmaceuticals by increasing the perception of sweet taste. Compoundswhich inhibit the activity of the T1R3 receptor may be used to block theperception of sweetness.

[0009] The invention is based, in part, on the discovery of a GPCRexpressed at high levels in taste receptor cells. In taste transduction,sweet compounds are thought to act via a second messenger cascadeutilizing PLCβ2 and IP₃. Co-localization of α-gustducin, PLCβ₂ Gβ3 andGγ13 and T1R3 to one subset of taste receptor cells indicates that theymay function in the same transduction pathway.

DEFINITIONS

[0010] As used herein, italicizing the name of T1R3 shall indicate theT1R3 gene, T1R3 DNA, cDNA, or RNA, in contrast to its encoded proteinproduct which is indicated by the name of T1R3 in the absence ofitalicizing. For example, “T1R3” shall mean the T1R3 gene, T1R3 DNA,cDNA, or RNA whereas “T1R3” shall indicate the protein product of theT1R3 gene.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1A. Synteny between human 1 p36.33 and mouse 4pterchromosomal regions near the mouse Sac locus. Shaded circles indicatethe approximate location of the predicted start codons for each gene;arrows indicate the full span of each gene including both introns andexons; arrowheads indicate the approximate location of eachpolyadenylation signal. Genes indicated by lowercase letters werepredicted by Genscan and named according to their closest homolog. Genesindicated by capital letters (T1R3 and DVL1) were experimentallyidentified and verified. The mouse marker D18346 indicated is closelylinked to the Sac locus and lies within the predicted pseudouridinesynthase-like gene. The region displayed corresponds to ˜45,000 bp; thebottom scale marker indicates kilobases (K).

[0012]FIG. 1B. The nucleotide and predicted amino acid sequences ofhuman T1R3. The ends of the introns are indicated in highlighted lowercase letters.

[0013]FIG. 1C. Predicted secondary structure of human T1R3. T1R3 ispredicted to have seven transmembrane helices and a large N-terminaldomain. Placement of the transmembrane segments was according to theTMpred program. Placement of the dimerization and ligand binding domain,and the cysteine-rich domain were based on the mGluR1 receptor and otherfamily 3 GPCRs (19).

[0014]FIG. 2A. Distribution of T1R3 mRNA in mouse tissues and mousetaste cells. Autoradiogram of a Northern blot hybridized with mouse T1R3cDNA. Each lane contained 25 μg of total RNA isolated from the followingmouse tissues: circumvallate and foliate papillae-enriched lingualtissue (Taste), lingual tissue devoid of taste buds (Non-Taste), brain,retina, olfactory epithelium (Olf Epi), stomach, small intestine (SmallInt), thymus, heart, lung, spleen, skeletal muscle (Ske Mus), liver,kidney, uterus and testis. A 7.2 kb transcript was detected only in thetaste tissue, and a slightly larger transcript was detected in testis.The blot was exposed to X-ray film for three days. The same blot wasstripped and reprobed with a β-actin cDNA (lower panel) and exposed forone day. The size of the RNA marker (in kilobases) is indicated in theright margin.

[0015]FIG. 2B. The genomic sequence of the Sac region from mouse wasused as a query to search the mouse expressed sequence tag (est)database. Matches to the est database are shown in solid red andindicate exons; gaps in a particular est match are shown by black hashedlines and indicate an intron. The clustered nature of the est matchesdemarcates the extent of each of the genes within this region. The nearabsence of ests at the position of T1R3 is consistent with the highlyrestricted pattern of expression seen in FIG. 2a.

[0016]FIG. 3A. T1R3 expression in taste receptor cells. Photomicrographsof frozen sections of mouse taste papillae hybridized with ³³P-labelledantisense RNA probes for T1R3 and α-gustducin. Bright-field images ofcircumvallate (a), foliate (b), and fungiform (c) papillae hybridized tothe antisense T1R3 probe demonstrate taste bud-specific expression ofT1R3. Control bright-field images of circumvallate (e), foliate (f), andfungiform papillae (g) hybridized to the sense T1R3 probe showed nononspecific binding. The level of expression and broad distribution ofT1R3 expression in taste buds was comparable to that of α-gustducin asshown in the bright field image of circumvallate papilla hybridized toantisense α-gustducin probe (d). The control bright field image ofcircumvallate papilla hybridized to the sense α-gustducin probe (h)showed no nonspecific binding.

[0017]FIG. 3B. Profiling the pattern of expression of T1R3, α-gustducin,Gγ13 and PLCβ2 in taste tissue and taste cells. Left panel: Southernhybridization to RT-PCR products from murine taste tissue (T) andcontrol non-taste lingual tissue (N). 3′-region probes from T1R3,α-gustducin (Gust), Gγ13, PLCβ2 and glyceraldehyde 3-phosphatedehydrogenase (G3PDH) were used to probe the blots. Note that T1R3,α-gustducin, Gγ13 and PLCβ2 were all expressed in taste tissue, but notin non-taste tissue. Right panel: Southern hybridization to RT-PCRproducts from 24 individually amplified taste receptor cells. 19 cellswere GFP-positive (+), 5 cells were GFP-negative (−). Expression ofα-gustducin, Gγ13 and PLCβ2 was fully coincident. Expression of T1R3overlapped partially with that of α-gustducin, Gγ13 and PLCβ2. G3PDHserved as a positive control to demonstrate successful amplification ofproducts.

[0018]FIG. 4. Co-localization of T1R3 PLCβ2 and α-gustducin in tastereceptor cells of human circumvallate papillae. (a, c) Longitudinalsections from human circumvallate papillae were labeled with rabbitantisera directed against a C-terminal peptide of human T1R3, along witha Cy3-conjugated anti-rabbit secondary antibody. (b) T1R3immunoreactivity in longitudinal sections from human papillae wasblocked by pre-incubation of the T1R3 antibody with the cognate peptide.(d) A longitutidinal section adjacent to that in sections of humanfungiform papillae double immunostained for T1R3 (h) and α-gustducin(i). The overlay of the two images is shown in (j). Magnification was200X (a-d) or 400X (e-j).

[0019]FIG. 5A. mT1R3 allelic differences. mT1R3 allelic differencesbetween eight inbred mouse strains. All non-taster strains showedidentical sequences and were grouped in one row. In the bottom row theamino acid immediately before the position number is always from thenon-tasters, while the amino acid immediately before the position numberis from whichever tasters differed at that position from thenon-tasters. The two columns in bold represent positions where alltasters differed from non-tasters and where the differences innucleotide sequence result in amino acid substitutions. Nucleotidedifferences that do not alter the encoded amino acid are indicated as s:silent. Nucleotide differences within introns are indicated as i:intron.

[0020]FIG. 5B. Genealogy of the inbred strains of mice analyzed in (a).The year in which the strains were developed is indicated betweenbrackets following the stain name. The laboratories in which these micewere established are indicated.

[0021]FIG. 6. The amino acid sequence of mouse T1R3 is aligned with thatof two other rat taste receptors (rT1R1 and rT1R2), the murineextracellular calcium sensing (mECaSR) and the metabotropic glutamatetype 1 (mGluR1) receptors. Regions of identity among all five receptorsare indicated by white letters on black; regions where one or more ofthese receptors share identity with T1R3 are indicated by black letterson gray. Boxes with dashed lines indicate regions predicted to beinvolved in dimerization (based upon the solved structure for the aminoterminal domain of mGluR1); filled circles indicate predicted ligandbinding residues based on mGluR1; blue lines linking cysteine residuesindicate predicted intermolecular disulfide bridges based on mGluR1.Amino acid sequences noted above the alignment indicate polymorphismsthat are found in all strains of nontaster mice. The predicted N-linkedglycosylation site conserved in all five receptors is indicated by ablack squiggle; the predicted N-linked glycosylation site specific toT1R3 in nontaster strains of mice is indicated by the red squiggle.

[0022]FIG. 7. The predicted three dimensional structure of theamino-terminal domain (ATD) of T1R3 modeled on that of mGluR1 (19) usingthe Modeller program. The model shows a homodimer of T1R3. (a) The viewfrom the “top” of the dimer looking down from the extracellular spacetoward the membrane. (b) The T1R3 dimer viewed from the side. In thisview the transmembrane region (not displayed) would attach to the bottomof the dimer. (c) The T1R3 dimer is viewed from the side as in (b),except the two dimers have been spread apart (indicated by the doubleheaded arrow) to reveal the contact surface. A space-fillingrepresentation (colored red) of three glycosyl moieties(N-acetyl-galactose-N-acetyl-galactose-Mannose) has been added at thenovel predicted site of glycosylation of non-taster mT1R3. Note that theaddition of even three sugar moieties at this site is stericallyincompatible with dimerization. Regions of T1R3 corresponding to thoseof mGluR1 involved in dimerization are shown by space filling aminoacids. The four different segments that form the predicted dimerizationsurface are color-coded in the same way as are the dashed boxes in FIG.5. The portions of the two molecules outside of the dimerization regionare represented by a backbone tracing. The two polymorphic amino acidresidues of T1R3 that differ in taster vs. non-taster strains of miceare within the predicted dimerization interface nearest the aminoterminus (colored light blue). The additional N-glycosylation site ataa58 unique to the non-taster form of T1R3 is indicated in each panel bythe straight arrows.

DETAILED DESCRIPTION OF THE INVENTION

[0023] T1R3 is a novel receptor that participates in receptor-mediatedtaste signal transduction and belongs to the family 3 G protein coupledreceptors. The present invention encompasses T1R3 nucleotides, T1R3proteins and peptides, as well as antibodies to the T1R3 protein. Theinvention also relates to host cells and animals genetically engineeredto express the T1R3 receptor or to inhibit or “knock-out” expression ofthe animal's endogenous T1R3.

[0024] The invention further provides screening assays. designed for theidentification of modulators, such as agonists and antagonists, of T1R3activity. The use of host cells that naturally express T1R3 orgenetically engineered host cells and/or animals offers an advantage inthat such systems allow the identification of compounds that affect thesignal transduced by the T1R3 receptor protein.

[0025] Various aspects of the invention are described in greater detailin the subsections below.

The T1R3 Gene

[0026] The cDNA sequence and deduced amino acid sequence of human T1R3is shown in FIG. 1B. The T1R3 nucleotide sequences of the inventioninclude: (a) the DNA sequence shown in FIG. 1B; (b) nucleotide sequencesthat encode the amino acid sequence shown in FIG. 1B; (c) any nucleotidesequence that (i) hybridizes to the nucleotide sequence set forth in (a)or (b) under stringent conditions, e.g., hybridization to filter-boundDNA in 0.5 M NaHPO₄, 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65EC,and washing in 0.1×SSC/0.1% SDS at 68EC (Ausubel F. M. et al., eds.,1989, Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3)and (ii) encodes a functionally equivalent gene product; and (d) anynucleotide sequence that hybridizes to a DNA sequence that encodes theamino acid sequence shown in FIG. 1B, under less stringent conditions,such as moderately stringent conditions, e.g., washing in 0.2×SSC/0.1%SDS at 42EC (Ausubel et al., 1989 supra), yet which still encodes afunctionally equivalent T1R3 gene product. Functional equivalents of theT1R3 protein include naturally occurring T1R3 present in species otherthan humans. The invention also includes degenerate variants ofsequences (a) through (d). The invention also includes nucleic acidmolecules, that may encode or act as T1R3 antisense molecules, useful,for example, in T1R3 gene regulation (for and/or as antisense primers inamplification reactions of T1R3 gene nucleic acid sequences).

[0027] In addition to the T1R3 nucleotide sequences described above,homologs of the T1R3 gene present in other species can be identified andreadily isolated, without undue experimentation, by molecular biologicaltechniques well known in the art. For example, cDNA libraries, orgenomic DNA libraries derived from the organism of interest can bescreened by hybridization using the nucleotides. described herein ashybridization or amplification probes.

[0028] The invention also encompasses nucleotide sequences that encodemutant T1R3s, peptide fragments of the T1R3, truncated T1R3, and T1R3fusion proteins. These include, but are not limited to nucleotidesequences encoding polypeptides or peptides corresponding to functionaldomains of T1R3, including but not limited to, the ATD (amino terminaldomain) that is believed to be involved in ligand binding anddimerization, the cysteine rich domain, and/or the transmembranespanning domains of T1R3, or portions of these domains; truncated T1R3sin which one or two domains of T1R3 is deleted, e.g., a functional T1R3lacking all or a portion of the ATD region. Nucleotides encoding fusionproteins may include but are not limited to full length T1R3, truncatedT1R3 or peptide fragments of T1R3 fused to an unrelated protein orpeptide such as an enzyme, fluorescent protein, luminescent protein,etc., which can be used as a marker.

[0029] Based on the model of T1R3's structure, it is predicted that T1R3dimerizes to form a functional receptor. Thus, certain of thesetruncated or mutant T1R3 proteins may act as dominant-negativeinhibitors of the native T1R3 protein. T1R3 nucleotide sequences may beisolated using a variety of different methods known to those skilled inthe art. For example, a cDNA library constructed using RNA from a tissueknown to express T1R3 can be screened using a labeled T1R3 probe.Alternatively, a genomic library may be screened to derive nucleic acidmolecules encoding the T1R3 receptor protein. Further, T1R3 nucleic acidsequences may be derived by performing PCR using two oligonucleotideprimers designed on the basis of the T1R3 nucleotide sequences disclosedherein. The template for the reaction may be cDNA obtained by reversetranscription of mRNA prepared from cell lines or tissue known toexpress T1R3.

[0030] The invention also encompasses (a) DNA vectors that contain anyof the foregoing T1R3 sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingT1R3 sequences operatively associated with a regulatory element thatdirects the expression of the T1R3 coding sequences; (c) geneticallyengineered host cells that contain any of the foregoing T1R3 sequencesoperatively associated with a regulatory element that directs theexpression of the T1R3 coding sequences in the host cell; and (d)transgenic mice or other organisms that contain any of the foregoingT1R3 sequences. As used herein, regulatory elements include but are notlimited to inducible and non-inducible promoters, enhancers, operatorsand other elements known to those skilled in the art that drive andregulate expression.

T1R3 Proteins and Polypeptides

[0031] T1R3 protein, polypeptides and peptide fragments, mutated,truncated or deleted forms of the T1R3 and/or T1R3 fusion proteins canbe prepared for a variety of uses, including but not limited to thegeneration of antibodies, the identification of other cellular geneproducts involved in the regulation of T1R3 mediated taste transduction,and the screening for compounds that can be used to modulate tasteperception such as novel sweetners and taste modifiers.

[0032]FIG. 1B shows the deduced amino acid sequence of the human T1R3protein. The T1R3 amino acid sequences of the invention include theamino acid sequence shown in FIG. 1B. Further, T1R3s of other speciesare encompassed by the invention. In fact, any T1R3 protein encoded bythe T1R3 nucleotide sequences described in Section 5.1, above, is withinthe scope of the invention.

[0033] The invention also encompasses proteins that are functionallyequivalent to the T1R3 encoded by the nucleotide sequences described inSection 5.1, as judged by any of a number of criteria, including but notlimited to the ability of a sweet tastant to activate T1R3 in a tastereceptor cell, leading to transmitter release from the taste receptorcell into the synapse and activation of an afferent nerve. Suchfunctionally equivalent T1R3 proteins include but are not limited toproteins having additions or substitutions of amino acid residues withinthe amino acid sequence encoded by the T1R3 nucleotide sequencesdescribed, above, in Section 5.1, but which result in a silent change,thus producing a functionally equivalent gene product.

[0034] Peptides corresponding to one or more domains of T1R3 (e.g.,amino terminal domain, the cysteine rich domain and/or the transmembranespanning domains), truncated or deleted T1R3s (e.g., T1R3 in which theamino terminal domain, the cysteine rich domain and/or the transmembranespanning domains is deleted) as well as fusion proteins in which thefull length T1R3, a T1R3 peptide or a truncated T1R3 is fused to anunrelated protein are also within the scope of the invention and can bedesigned on the basis of the T1R3 nucleotide and T1R3 amino acidsequences disclosed herein. Such fusion proteins include fusions to anenzyme, fluorescent protein, or luminescent protein which provide amarker function.

[0035] While the T1R3 polypeptides and peptides can be chemicallysynthesized (e.g., see Creighton, 1983, Proteins: Structures andMolecular Principles, W. H. Freeman & Co., N.Y.), large polypeptidesderived from T1R3 and the full length T1R3 itself may be advantageouslyproduced by recombinant DNA technology using techniques well known inthe art for expressing a nucleic acid containing T1R3 gene sequencesand/or coding sequences. Such methods can be used to constructexpression vectors containing the T1R3 nucleotide sequences described inSection 5.1 and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination.(See, for example, the techniques described in Sambrook et al., 1989,supra, and Ausubel et al., 1989, supra).

[0036] A variety of host-expression vector systems may be util ized toexpress the T1R3 nucleotide sequences of the invention. Where the T1R3peptide or polypeptide is expressed as a soluble derivative (e.g.,peptides corresponding to the amino terminal domain the cysteine richdomain and/or the transmembrane spanning domain) and is not secreted,the peptide or polypeptide can be recovered from the host cell.Alternatively, where the T1R3 peptide or polypeptide is secreted thepeptide or polypeptides may be recovered from the culture media.However, the expression systems also include engineered host cells thatexpress T1R3 or functional equivalents, anchored in the cell membrane.Purification or enrichment of the T1R3 from such expression systems canbe accomplished using appropriate detergents and lipid micelles andmethods well known to those skilled in the art. Such engineered hostcells themselves may be used in situations where it is important notonly to retain the structural and functional characteristics of theT1R3, but to assess biological activity, i.e., in drug screening assays.

[0037] The expression systems that may be used for purposes of theinvention include but are not limited to microorganisms such as bacteriatransformed with recombinant bacteriophage, plasmid or cosmid DNAexpression vectors containing T1R3 nucleotide sequences; yeasttransformed with recombinant yeast expression vectors containing T1R3nucleotide sequences or mammalian cell systems harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells or from mammalian viruses.

[0038] Appropriate expression systems can be chosen to ensure that thecorrect modification, processing and sub-cellular localization of theT1R3 protein occurs. To this end, eukaryotic host cells which possessthe ability to properly modify and process the T1R3 protein arepreferred. For long-term, high yield production of recombinant T1R3protein, such as that desired for development of cell lines forscreening purposes, stable expression is preferred. Rather than usingexpression vectors which contain origins of replication, host cells canbe transformed with DNA controlled by appropriate expression controlelements and a selectable marker gene, i.e., tk, hqprt, dhfr, neo, andhygro gene, to name a few. Following the introduction of the foreignDNA, engineered cells may be allowed to grow for 1-2 days in enrichedmedia, and then switched to a selective media. Such engineered celllines may be particularly useful in screening and evaluation ofcompounds that modulate the endogenous activity of the T1R3 geneproduct.

Transgenic Animals

[0039] The T1R3 gene products can also be expressed in transgenicanimals. Animals of any species, including, but not limited to, mice,rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-humanprimates, e.g., baboons, monkeys, and chimpanzees may be used togenerate T1R3 transgenic animals.

[0040] Any technique known in the art may be used to introduce the T1R3transgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci. USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell,56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

[0041] The present invention provides for transgenic animals that carrythe T1R3 transgene in all their cells, as well as animals which carrythe transgene in some, but not all their cells, i.e., mosaic animals.The transgene may also be selectively introduced into and activated in aparticular cell type by following, for example, the teaching of Lasko etal., (Lasko, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236).The regulatory sequences required for such a cell-type specificactivation will depend upon the particular cell type of interest, andwill be apparent to those of skill in the art. When it is desired thatthe T1R3 transgene be integrated into the chromosomal site of theendogenous T1R3 gene, gene targeting is preferred. Briefly, when such atechnique is to be utilized, vectors containing some nucleotidesequences homologous to the endogenous T1R3 gene are designed for thepurpose of integrating, via homologous recombination with chromosomalsequences, into and disrupting the function of the nucleotide sequenceof the endogenous T1R3 gene.

[0042] Once transgenic animals have been generated, the expression ofthe recombinant T1R3 gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include but are not limited to Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of T1R3 gene-expressing tissue may also beevaluated immunocytochemically using antibodies specific for the T1R3transgene product.

Antibodies to T1R3 Proteins

[0043] Antibodies that specifically recognize one or more epitopes ofT1R3, or epitopes of conserved variants of T1R3, or peptide fragments ofT1R3 are also encompassed by the invention. Such antibodies include butare not limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

[0044] The antibodies of the invention may be used, for example, inconjunction with compound screening schemes, as described, below, inSection 5.5, for the evaluation of the effect of test compounds onexpression and/or activity of the T1R3 gene product.

[0045] For production of antibodies, various host animals may beimmunized by injection with a T1R3 protein, or T1R3 peptide. Such hostanimals may include but are not limited to rabbits, mice, and rats, toname but a few. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.

[0046] Polyclonal antibodies comprising heterogeneous populations ofantibody molecules, may be derived from the sera of the immunizedanimals. Monoclonal antibodies may be obtained by any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique of Kohler and Milstein, (1975, Nature 256:495-497; and U.S.Pat. No. 4,376,110), the human B-cell hybridoma technique (Kosbor etal., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad.Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al.,1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgS, IgA, IgD and any subclasses thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titres of Mabs in vivo makes this the presentlypreferred method of production.

[0047] In addition, techniques developed for the production of “chimericantibodies” by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used(Morrison et al., 1984, Proc. Nat'l. Acad. Sci., 81:6851-6855; Neubergeret al., 1984, Nature, 312: 604-608; Takeda et al. 1985, Nature 314:452-454). Alternatively, techniques developed for the production ofhumanized antibodies (U.S. Pat. No. 5,585,089) or single chainantibodies (U.S. Pat. No. 4,946,778 Bird, 1988, Science 242: 423-426;Huston et al., 1988, Proc. Nat'l. Acad. Sci USA, 85: 5879-5883; and Wardet al., 1989, Nature 334: 544-546) may be used to produce antibodiesthat specifically recognize one or more epitopes of T1R3.

Screening Assays for Drugs and Other Chemical Compounds Useful inRegulation of Taste Perception

[0048] The present invention relates to screening assay systems designedto identify compounds or compositions that modulate T1R3 activity orT1R3 gene expression, and thus, may be useful for modulation of sweettaste perception.

[0049] In accordance with the invention, a cell-based assay system canbe used to screen for compounds that modulate the activity of the T1R3and thereby, modulate the perception of sweetness. To this end, cellsthat endogenously express T1R3 can be used to screen for compounds.Alternatively, cell lines, such as 293 cells, COS cells, CHO cells,fibroblasts, and the like, genetically engineered to express T1R3 can beused for screening purposes. Preferably, host cells geneticallyengineered to express a functional T1R3 are those that respond toactivation by sweet tastants, such as taste receptor cells. Further,ooyctes or liposomes engineered to express T1R3 may be used in assaysdeveloped to identify modulators of T1R3 activity.

[0050] The present invention provides for methods for identifying acompound that induces the perception of a sweet taste (a “sweetnessactivator”) comprising (i) contacting a cell expressing the T1R3receptor with a test compound and measuring the level of T1R3activation; (ii) in a separate experiment, contacting a cell expressingthe T1R3 receptor protein with a vehicle control and measuring the levelof T1R3 activation where the conditions are essentially the same as inpart (i), and then (iii) comparing the level of activation of T1R3measured in part (i) with the level of activation of T1R3 in part (ii),wherein an increased level of activated T1R3 in the presence of the testcompound indicates that the test compound is a T1R3 activator.

[0051] The present invention also provides for methods for identifying acompound that inhibits the perception of a sweet taste (a “sweetnessinhibitor”) comprising (i) contacting a cell expressing the T1R3receptor protein with a test compound in the presence of a sweet tastantand measuring the level of T1R3 activation; (ii) in a separateexperiment, contacting a cell expressing the T1R3 receptor protein witha sweet tastant and measuring the level of T1R3 activation, where theconditions are essentially the same as in part (i) and then (iii)comparing the level of activation of T1R3 measured in part (i) with thelevel of activation of T1R3 in part (ii), wherein a decrease level ofactivation of T1R3 in the presence of the test compound indicates thatthe test compound is a T1R3 inhibitor.

[0052] A “sweet tastant”, as defined herein, is a compound or molecularcomplex that induces, in a subject, the perception of a sweet taste. Inparticular, a sweet tastant is one which results in the activation ofthe T1R3 protein resulting in one or more of the following:. (i) aninflux of Ca⁺² into the cell; (ii) release of Ca⁺² from internal stores;(iii) activation of coupled G proteins such as Gs and/or gustducin; (iv)activation of secon messenger-regulating enzymes such as adenylylcyclase and/or phospholipase C. Examples of sweet tastants include butare not limited to saccharin or sucrose, or other sweetners.

[0053] In utilizing such cell systems, the cells expressing the T1R3receptor are exposed to a test compound or to vehicle controls (e.g.,placebos). After exposure, the cells can be assayed to measure theexpression and/or activity of components of the signal transductionpathway of T1R3, or the activity of the signal transduction pathwayitself can be assayed.

[0054] The ability of a test molecule to modulate the activity of T1R3may be measured using standard biochemical and physiological techniques.Responses such as activation or suppression of catalytic activity,phosphorylation or dephosphorylation of T1R3 and/or other proteins,activation or modulation of second messenger production, changes incellular ion levels, association, dissociation or translocation ofsignaling molecules, or transcription or translation of specific genesmay be monitored. In non-limiting embodiments of the invention, changesin intracellular Ca²⁺ levels may be monitored by the fluorescence ofindicator dyes such as indo, fura, etc. Additionally, changes in cAMP,cGMP, IP₃, and DAG levels may be assayed. In yet another embodiment,activation of adenylyl cyclase, guanylyl cyclase, protein kinase A andCa²⁺ sensitive release of neurotransmitters may be measured to identifycompounds that modulate T1R3 signal transduction. Further, changes inmembrane potential resulting from modulation of the T1R3 channel proteincan be measured using a voltage clamp or patch recording methods. In yetanother embodiment of the invention, a microphysiometer can be used tomonitor cellular activity.

[0055] For example, after exposure to a test compound, cell lysates canbe assayed for increased intracellular levels of Ca²⁺ and activation ofcalcium dependent downstream messengers such as adenylyl cyclase,protein kinase A or cAMP. The ability of a test compound to increaseintracellular levels of Ca²⁺, activate protein kinase A or increase cAMPlevels compared to those levels seen with cells treated with a vehiclecontrol, indicates that the test compound acts as an agonist (i.e., is aT1R3 activator) and induces signal transduction mediated by the T1R3expressed by the host cell. The ability of a test compound to inhibitsweet tastant induced calcium influx, inhibit protein kinase A ordecrease cAMP levels compared to those levels seen with a vehiclecontrol indicates that the test compound acts as an antagonist (i.e., isa T1R3 inhibitor) and inhibits signal transduction mediated by T1R3.

[0056] In a specific embodiment of the invention, levels of cAMP can bemeasured using constructs containing the cAMP responsive element linkedto any of a variety of different reporter genes. Such reporter genes mayinclude but are not limited to chloramphenicol acetyltransferase (CAT),luciferase, β-glucuronidase (GUS), growth hormone, or placental alkalinephosphatase (SEAP). Such constructs are introduced into cells expressingT1R3 thereby providing a recombinant cell useful for screening assaysdesigned to identify modulators of T1R3 activity.

[0057] Following exposure of the cells to the test compound, the levelof reporter gene expression may be quantitated to determine the testcompound's ability to regulate T1R3 activity. Alkaline phosphataseassays are particularly useful in the practice of the invention as theenzyme is secreted from the cell. Therefore, tissue culture supernatantmay be assayed for secreted alkaline phosphatase. In addition, alkalinephosphatase activity may be measured by calorimetric, bioluminescent orchemilumenscent assays such as those described in Bronstein, I. et al.(1994, Biotechnicues 17: 172-177). Such assays provide a simple,sensitive easily automatable detection system for pharmaceuticalscreening.

[0058] Additionally, to determine intracellular cAMP concentrations, ascintillation proximity assay (SPA) may be utilized (SPA kit is providedby Amersham Life Sciences, Illinois). The assay utilizes ¹²⁵I-labelcAMP, an anti-cAMP antibody, and a scintillant-incorporated microspherecoated with a secondary antibody. When brought into close proximity tothe microsphere through the labeled cAMP-antibody complex, ¹²⁵I willexcite the scintillant to emit light. Unlabeled cAMP extracted fromcells competes with the ²⁵I-labeled cAMP for binding to the antibody andthereby diminishes scintillation. The assay may be performed in 96-wellplates to enable high-throughput screening and 96 well-basedscintillation counting instruments such as those manufactured by Wallacor Packard may be used for readout.

[0059] In yet another embodiment of the invention, levels ofintracellular Ca²⁺ can be monitored using Ca²⁺ indication dyes, such asFluo-3 and Fura-Red using methods such as those described in Komuro andRakic, 1998, In: The Neuron in Tissue Culture. L. W. Haymes, Ed. Wiley,New York.

[0060] Test activators which activate the activity of T1R3, identifiedby any of the above methods, may be subjected to further testing toconfirm their ability to induce a sweetness perception. Test inhibitorswhich inhibit the activation of T1R3 by sweet tastants, identified byany of the above methods, may then be subjected to further testing toconfirm their inhibitory activity. The ability of the test compound tomodulate the activity of the T1R3 receptor may be evaluated bybehavioral, physiologic, or in vitro methods.

[0061] For example, a behavioral study may be performed where a testanimal may be offered the choice of consuming a composition comprisingthe putative T1R3 activator and the same composition without the addedcompound. A preference for the composition comprising a test compound,indicated, for example, by greater consumption, would have a positivecorrelation with activation of T1R3 activity. Additionally, lack ofpreference by a test animal of food containing a putative inhibitor ofT1R3 in the presence of a sweetner would have a positive correlationwith the identification of an sweetness inhibitor.

[0062] In addition to cell based assays, non-cell based assay systemsmay be used to identify compounds that interact with, e.g., bind toT1R3. Such compounds may act as antagonists or agonists of T1R3 activityand may be used to regulate sweet taste perception.

[0063] To this end, soluble T1R3 may be recombinantly expressed andutilized in non-cell based assays to identify compounds that bind toT1R3. The recombinantly expressed T1R3 polypeptides or fusion proteinscontaining one or more of the domains of T1R3 prepared as described inSection 5.2, infra, can be used in the non-cell based screening assays.For example, peptides corresponding to the amino terminal domain that isbelieved to be involved in ligand binding and dimerization, the cysteinerich domain and/or the transmembrane spanning domains of T1R3, or fusionproteins containing one or more of the domains of T1R3 can be used innon-cell based assay systems to identify compounds that bind to aportion of the T1R3; such compounds may be useful to modulate the signaltransduction pathway of the T1R3. In non-cell based assays therecombinantly expressed T1R3 may be attached to a solid substrate suchas a test tube, microtitre well or a column, by means well known tothose in the art (see Ausubel et al., supra) . The test compounds arethen assayed for their ability to bind to the T1R3.

[0064] The T1R3 protein may be one which has been fully or partiallyisolated from other molecules, or which may be present as part of acrude or semi-purified extract. As a non-limiting example, the T1R3protein may be present in a preparation of taste receptor cellmembranes. In particular embodiments of the invention, such tastereceptor cell membranes may be prepared as set forth in Ming, D. et al.,1998, Proc. Natl. Sci. U.S.A. 95:8933-8938, incorporated by referenceherein. Specifically, bovine circumvallate papillae (“taste tissue”,containing taste receptor cells), may be hand dissected, frozen inliquid nitrogen, and stored at −80EC prior to use. The collected tissuesmay then be homogenized with a Polytron homogenizer (three cycles of 20seconds each at 25,000 RPM) in a buffer containing 10 mM Tris at pH 7.5,10% vol/vol glycerol, 1 mM EDTA, 1 mM DTT, 10 μg/μl pepstatin A, 10μg/μl leupeptin, 10 μg/μl aprotinin, and 100 μM 4-(2-amino ethyl)benzenesulfoyl fluoride hydrochloride. After particulate removal bycentrifugation at 1,500×g for 10 minutes, taste membranes may becollected by centrifugation at 45,000×g for 60 minutes. The pelletedmembranes may then be rinsed twice, re-suspended in homogenizationbuffer lacking protease inhibitors, and further homogenized by 20passages through a 25 gauge needle. Aliquots may then be either flashfrozen or stored on ice until use. As another non-limiting example, thetaste receptor may be derived from recombinant clones (see Hoon, M. R.et al., 1999 Cell 96, 541-551).

[0065] Assays may also be designed to screen for compounds that regulateT1R3 expression at either the transcriptional or translational level. Inone embodiment, DNA encoding a reporter molecule can be linked to aregulatory element of the T1R3 gene and used in appropriate intactcells, cell extracts or lysates to identify compounds that modulate T1R3gene expression. Appropriate cells or cell extracts are prepared fromany cell type that normally expresses the T1R3 gene, thereby ensuringthat the cell extracts contain the transcription factors required for invitro or in vivo transcription. The screen can be used to identifycompounds that modulate the expression of the reporter construct. Insuch screens, the level of reporter gene expression is determined in thepresence of the test compound and compared to the level of expression inthe absence of the test compound.

[0066] To identify compounds that regulate T1R3 translation, cells or invitro cell lysates containing T1R3 transcripts may be tested formodulation of T1R3 mRNA translation. To assay for inhibitors of T1R3translation, test compounds are assayed for their ability to modulatethe translation of T1R3 mRNA in in vitro translation extracts.

[0067] In addition, compounds that regulate T1R3 activity may beidentified using animal models. Behavioral, physiological, orbiochemical methods may be used to determine whether T1R3 activation hasoccurred. Behavioral and physiological methods may be practiced in vivo.As an example of a behavioral measurement, the tendency of a test animalto voluntarily ingest a composition, in the presence or absence of testactivator, may be measured. If the test activator induces T1R3 activityin the animal, the animal may be expected to experience a sweet taste,which would encourage it to ingest more of the composition. If theanimal is given a choice of whether to consume a composition containinga sweet tastant only (which activates T1R3) or a composition containinga test inhibitor together with a sweet tastant, it would be expected toprefer to consume the composition containing sweet tastant only. Thus,the relative preference demonstrated by the animal inversely correlateswith the activation of the T1R3 receptor.

[0068] Physiological methods include nerve response studies, which maybe performed using a nerve operably joined to a taste receptor cellcontaining tissue, in vivo or in vitro. Since exposure to sweet tastantwhich results in T1R3 activation may result in an action potential intaste receptor cells that is then propagated through a peripheral nerve,measuring a nerve response to a sweet tastant is, inter alia, anindirect measurement of T1R3 activation. An example of nerve responsestudies performed using the glossopharyngeal nerve are described inNinomiya, Y., et al., 1997, Am. J. Physiol. (London) 272:R1002-R1006.

[0069] The assays described above can identify compounds which modulateT1R3 activity. For example, compounds that affect T1R3 activity includebut are not limited to compounds that bind to the T1R3, and eitheractivate signal transduction (agonists) or block activation(antagonists). Compounds that affect T1R3 gene activity (by affectingT1R3 gene expression, including molecules, e.g., proteins or smallorganic molecules, that affect transcription or interfere with spacingevents so that expression of the full length or the truncated form ofthe T1R3 can be modulated) can also be identified using the screens ofthe invention. However, it should be noted that the assays described canalso identify compounds that modulate T1R3 signal transduction (e.g.,compounds which affect downstream signaling events, such as inhibitorsor enhancers of G protein activities which participate in transducingthe signal activated by tastants binding to their receptor). Theidentification and use of such compounds which affect signaling eventsdownstream of T1R3 and thus modulate effects of T1R3 on the perceptionof taste are within the scope of the invention.

[0070] The compounds which may be screened in accordance with theinvention include, but are not limited to, small organic or inorganiccompounds, peptides, antibodies and fragments thereof, and other organiccompounds (e.g., peptidomimetics) that bind to T1R3 and either mimic theactivity triggered by the natural tastant ligand (i.e., agonists) orinhibit the activity triggered by the natural ligand (i.e.,antagonists). Such compounds may be naturally occurring compounds suchas those present in fermentation broths, cheeses, plants, and fungi, forexample.

[0071] Compounds may include, but are not limited to, peptides such as,for example, soluble peptides, including but not limited to members ofrandom peptide libraries (see, e.g., Lam, K. S. et al., 1991, Nature354:82-84; Houghten, R. et al., 1991, Nature 354:84-86); andcombinatorial chemistry—derived molecular library made of D- and/orL-configuration amino acids, phosphopeptides (including, but not limitedto, members of random or partially degenerate, directed phosphopeptidelibraries; (see, e.g., Songyang, Z. et al., 1993, Cell 72:767-778),antibodies (including, but not limited to, polyclonal, monoclonal,humanized, anti-idiotypic, chimeric or single chain antibodies, and FAb,F(ab′)₂ and FAb expression library fragments, and epitope bindingfragments thereof), and small organic or inorganic molecules.

[0072] Other compounds which may be screened in accordance with theinvention include but are not limited to small organic molecules thataffect the expression of the T1R3 gene or some other gene involved inthe T1R3 signal transduction pathway (e.g., by interacting with theregulatory region or transcription factors involved in gene expression);or such compounds that affect the activity of the T1R3 or the activityof some other intracellular factor involved in the T1R3 signaltransduction pathway, such as, for example, a T1R3 associated G-protein.

Compositions Containing Modulators of T1R3 and Their Uses

[0073] The present invention provides for methods of inducing a sweettaste resulting from contacting a taste tissue of a subject with a sweettastant, comprising administering to the subject an effective amount ofa T1R3 activator, such as a T1R3 activator identified by measuring T1R3activation as set forth in Section 5.5 supra. The present invention alsoprovides for methods of inhibiting the sweet taste of a composition,comprising incorporating, in the composition, an effective amount of aT1R3 inhibitor. An “effective amount” of the T1R3 inhibitor is an amountthat subjectively decreases the perception of sweet taste and/or that isassociated with a detectable decrease in T1R3 activation as measured byone of the above assays.

[0074] The present invention further provides for a method of producingthe perception of a sweet taste by a subject, comprising administering,to the subject, a composition comprising a compound that activates T1R3activity such as a sweetness activator identified as set forth inSection 5.5 supra. The composition may comprise an amount of activatorthat is effective in producing a taste recognized as sweet by a subject.

[0075] Accordingly, the present invention provides for compositionscomprising sweetness activators and sweetness inhibitors. Suchcompositions include any substances which may come in contact with tastetissue of a subject, including but not limited to foods, beverages,pharmaceuticals, dental products, cosmetics, and wetable glues used forenvelopes and stamps.

[0076] In one set of embodiments of the invention, T1R3 activators areutilized as food or beverage sweetners. In such instances, the T1R3activators of the invention are incorporated into foods or beverages,thereby enhancing the sweet flavor of the food or beverage withoutincreasing the carbohydrate content of the food.

[0077] In another embodiment of the invention, a sweetness activator isused to counteract the perception of bitterness associated with aco-present bitter tastant. In these embodiments, a composition of theinvention comprises a bitter tastant and a sweetness activator, wherethe sweetness activator is present at a concentration which inhibitsbitter taste perception. For example, when the concentration of bittertastant in the composition and the concentration of sweetness activatorin the composition are subjected to an assay as disclosed in Section 5.1supra.

[0078] The present invention may be used to improve the taste of foodsby increasing the perception of sweetness or by decreasing oreliminating the aversive effects of bitter tastants. If a bitter tastantis a food preservative, the T1R3 activators of the invention may permitor facilitate its incorporation into foods, thereby improving foodsafety. For foods administered as nutritional supplements, theincorporation of T1R3 activators of the invention may encourageingestion, thereby enhancing the effectiveness of these compositions inproviding nutrition or calories to a subject.

[0079] The T1R3 activators of the invention may be incorporated intomedical and/or dental compositions. Certain compositions used indiagnostic procedures have an unpleasant taste, such as contrastmaterials and local oral anesthetics. The T1R3 activators of theinvention may be used to improve the comfort of subjects undergoing suchprocedures by improving the taste of compositions. In addition, the T1R3activators of the invention may be incorporated into pharmaceuticalcompositions, including tablets and liquids, to improve their flavor andimprove patient compliance (particularly where the patient is a child ora non-human animal).

[0080] The T1R3 activators of the invention may be comprised incosmetics to improve their taste features. For example, but not by wayof limitation, the T1R3 activators of the invention may be incorporatedinto face creams and lipsticks. In addition, the T1R3 activators of theinvention may be incorporated into compositions that are not traditionalfoods, beverages, pharmaceuticals, or cosmetics, but which may contacttaste membranes. Examples include, but are not limited to, soaps,shampoos, toothpaste, denture adhesive, glue on the surfaces of stampsand envelopes, and toxic compositions used in pest control (e.g., rat orcockroach poison).

EXAMPLE Cloning and Characterization of the T1R3 Gene

[0081] The data presented below describes the identification of a noveltaste receptor, T1R3, as being Sac. This identification is based on thefollowing observations. T1R3 is the only GPCR present in a 1 million bpregion of human genomic DNA centered on the D18346 marker most tightlylinked to Sac. Expression of T1R3 is narrowly restricted and is highlyexpressed in a subset of taste receptor cells. Expression of T1R3 intaste receptor cells overlaps in large part with known and proposedelements of sweet transduction pathways (i.e. α-gustducin, Gγ13. T1R3 isa family 3 GPCR with a large extracellular domain sensitive to proteases(a known property of the sweet receptor). Most tellingly, a polymorphismin T1R3 was identified that differentiated all taster strains of micefrom all non-taster strains: T1R3 from non-tasters is predicted tocontain an N-terminal glycosylation site that based on modeling ofT1R3's structure would be expected to interfere with its dimerization.Hence, not only is T1R3 identified as sac, but based on the model ofT1R3 and this polymorphic change it is also likely to be asweet-responsive (i.e. sweet-liganded) taste receptor.

Gene Identification

[0082] To identify the mouse gene (pseudouridine synthase-like)containing the D18346 marker the D18346 sequence was used as a querysequence in a BlastN screen of the mouse expressed sequence tag (est)database. Each resulting overlapping sequence match was used iterativelyto extend the sequence until the nearly full length gene was determined.The resulting contig was translated and the predicted open reading framewas used as a query in a TBlastN search of the High Throughput GenomicSequence (HTGS) database. This search located a human BAC clone AL139287containing the human ortholog. Genscan was used to predict genes andexons in this clone. BlastN or TBlastN searches of either the NR or theest databases were used to further define known or unknown genes in thisand other clones. Each resulting predicted gene was used in TBlastN orBlastN searches of the HTGS to find overlapping BAC or PAC clones. Eachof the overlapping sequences was used in BlastN searches of the HTGS tocontinue the build of an unordered contig of the region. The predictedgenes and exons that resulted from this search were used to partiallyorder over 1 million bases of genomic sequence centered on thepseudouridine synthase-like gene containing the D18346 marker. Two humanclones were found to contain T1R3, the aforementioned AL139287 andAC026283. The human T1R3 gene was first predicted by Genscan andsubsequently confirmed by RT-PCR of human fungiform taste bud RNA and/orscreening of a human taste library. In addition to the abovemanipulations and searches we used an algorithm (designed to recognizetransmembrane spans in genomic sequence) to search all of the humangenomic clones on the p arm of human chromosome 1 from 1pter to 1p33(Sanger Center chromosome 1 mapping project, FC and HW, unpublished).This screen predicted T1R3 as well as T1R1 and T1R2. Human T1R3 lieswithin 20,000 bp of the D18346 marker and the pseudouridinesynthase-like gene and is the only predicted GPCR in this 1 million bpregion.

[0083] The human predicted gene was then used in a TBlastN screen of theCelera mouse fragment genomic database. Each matching fragment was usedto fill gaps and further extend the mouse T1R3 ortholog in repeatedBlastN searches. The following mouse fragments were used to build andrefine the mouse T1R3 genomic sequence: GA_(—)49588987, GA_(—)72283785,GA_(—)49904613, GA_(—)50376636, GA_(—)74432413, GA_(—)70914196,GA_(—)62197520, GA_(—)77291497, GA_(—)74059038, GA_(—)66556470,GA_(—)70030888, GA_(—)50488116, GA_(—)50689730, GA_(—)72936925,GA_(—)72154490, GA_(—)69808702. Genscan was used to predict the mousegene from the resulting genomic contig. The predicted mouse T1R3 genewas confirmed by RT-PCR of mouse taste bud RNA. Other genes from thehuman genomic region centered on D18346 were used to search the Celeramouse fragments database. The sequences from these searches were used tobuild a mouse genomic contig of this region and confirm the linkage ofD18346 with T1R3 in the mouse genome and the micro-synteny of the humanand mouse genes in this region. One gap in the genomic sequence, betweenthe 5′-end of T1R3 and the 3′-end of the glycolipid-transferase-likegene was bridged by PCR and confirmed by sequence analysis.

Northern Hybridization

[0084] Total RNAs were isolated from several mouse tissues using theTrizol reagents, then 25 μg of each RNA was electrophoresed per lane ona 1.5% agarose gel containing 6.7% formaldehyde. The samples weretransferred and fixed to a nylon membrane by UV irradiation. The blotwas prehybridized at 65° C. in 0.25 M sodium phosphate buffer (pH 7.2)containing. 7% SDS and 40 μg/ml herring sperm DNA with agitation for 5hours; hybridization for 20 hours with the ³²p -radiolabeled mouse T1R3probe was carried out in the same solution. The membrane was washedtwice at 65° C. in 20 mM sodium phosphate buffer (pH 7.2) containing 5%SDS for 40 minutes, twice at 65° C. in the same buffer containing 1% SDSfor 40 minutes, and once at 70° C. in 0.1×SSC and 0.1% SDS for 30minutes. The blot was exposed to X-ray film for 3 days at −80° C. withdual intensifying screens. The ³²p-labeled T1R3 probe was generated byrandom nonamer priming of a 1.34-kb cDNA fragment of murine T1R3corresponding to the 5′-end coding sequence using Exo(−) Klenowpolymerease in the presence of (α-³²P)-dCTP.

In Situ Hybridization

[0085] 33P-labeled RNA probes T1R3 (2.6 kb) and α-gustducin (1 kb)] wereused for in situ hybridization of frozen sections (10 μm) of mouselingual tissue. Hybridization and washing were as described (2). Slideswere coated with Kodak NTB-2 nuclear track emulsion and exposed at 4° C.for 3 weeks and then developed and fixed.

Gene Expression Profiling

[0086] Single taste receptor cell RT-PCR products (5 μl) werefractionated by size on a 1.6% agarose gel and transferred onto a nylonmembrane. The expression patterns of the isolated cells were determinedby Southern hybridization with 3′-end cDNA probes for mouse T1R3,α-gustducin, Gγ13, PLCβ2 and G3PDH. Blots were exposed for five hours at−80° C. Total RNAs from a single circumvallate papilla and asimilar-sized piece of non-gustatory epithelium were also isolated,reverse transcribed, amplified and analyzed as for the individual cells.

Immunocytochemistry

[0087] Polyclonal antisera against a hemocyanin-conjugated T1R3 peptide(T1R3-A, aa 829-843) were raised in rabbits. The PLC β2 antibody wasobtained from Santa-Cruz Biotechnologies. Ten micron thick frozensections of human lingual tissue (previously fixed in 4%paraformaldehyde and cryoprotected in 20% sucrose) were blocked in 3%BSA, 0.3% Triton X-100, 2% goat serum and 0.1% Na Azide in PBS for 1hour at room temperature and then incubated for 8 hours at 4° C. withpurified antibody against α-gustducin, or antiserum against T1R3(1:800). The secondary antibodies were Cy3-conjugated goat-anti-rabbitIg for T1R3 and fluorescein-conjugated goat-anti-rabbit Ig for PLC β2.PLC β2 and T1R3 immunoreactivities were blocked by preincubation of theantisera with the corresponding synthetic peptides at 10 μM and 20 μM,respectively. Preimmune serum did not show any immunoreactivity. Somesections were double-immunostained with T1R3 and PLC β2 antisera asdescribed (46). Briefly, sections were incubated sequentially with T1R3antiserum, anti-rabbit-Ig-Cy3 conjugate, normal anti-rabbit-Ig, PLCβ2antibody and finally with anti-rabbit-Ig-FITC conjugate withintermittent washes between each step. Control sections that wereincubated with all of the above except PLCβ2 antibody did not show anyfluorescence in the green channel.

Identification of Sequence Polymorphisms in mT1R3

[0088] Based on the sequence of mouse T1R3 obtained from the Celeramouse fragments database, oligonucleotide primers were designed toamplify DNA encoding regions with open reading frames. Total RNAisolated from taste papillae or tail genomic DNA isolated from onetaster (C57BL6J) and one non-taster (129/Svev) mouse strain each wereused as templates to amplify mouse T1R3 cDNA and genomic DNA usingRT-PCR and PCR, respectively. PCR products were sequenced completely inan ABI 310 automated sequencer. Based on the sequence obtained, foursets of oligonucleotide primers were used to amplify the T1R3 regionswhere polymorphisms were found between the two strains of mice. GenomicDNA from mouse strains DBA/2, BALB/c, C3H/HeJ, SWR and FVB/N, was usedas template. The amplicons were purified and directly sequenced. Thegenealogical tree of these strains of mice was based on Hogan et al,(47) and the Jackson laboratory web site (http://www.jax.org).

Modeling the Structure of T1R3

[0089] The amino terminal domains (ATDs) of mouse T1R3 and mouse GluR1were aligned using the ClustalW program (48). The alignment was manuallyedited to generate an optimal alignment based on structural andfunctional considerations. Atomic coordinates of the mGluR1 ATD crystalstructure (19) were obtained from the protein database and were usedalong with the alignment as the source of spatial restraints formodeling. The structural model of mouse T1R3 was generated using theprogram MODELLER (49). The original images for FIG. 7 were created usingthe programs Insight II and Weblab Viewer (Molecular Simulations Inc.)and then imported into Photoshop where the open view was created and thelabels were added.

Results Mapping of the Murine and Human Human Sac Regions

[0090] The murine Sac gene is the primary determinant of inter-strainpreference responses to sucrose, saccharin, acesulfame, dulcin, glycineand other sweeteners (9-12), however, the molecular nature of the Sacgene product is unknown. Taster vs. non-taster strains of mice displaydifferences in the electrophysiological responses of their taste nervesto sweeteners and sweet amino acids, arguing that Sac exerts its effecton the sweet pathway at the periphery (14, 18). The most likelyexplanation for these differences is an allelic difference in a geneencoding a sweet-responsive taste transduction element such as areceptor, G protein subunit, effector enzyme or other member of thesweet signaling pathway. It had been speculated that the Sac geneproduct modified a sweet-responsive receptor (12), was itself a tastereceptor (17) or a G protein subunit (14). As a first step towardidentifying the nature of the Sac gene we generated a contiguous map ofthe human genome in this region was generated. Starting with the mousemarker D18346 (16), which maps most closely to the sac locus at 4pter, anovel mouse gene from the est database was identified: D18346 is foundin the 3′ untranslated region (UTR) of a novel mouse gene with homologyto pseudouridine synthase. At the time this work was initiated thesequence of the human genome was nearly complete (although onlypartially assembled), while that of mouse was quite incomplete, hence,finished human genomic sequences and unfinished sequences from bacterialartificial chromosome (BAC) and P1 artificial chromosome (PAC) clonesknown to map to human chromosome 1pter-1p36.33 (syntenic to mouse 4pter)was screened for the ortholog of the novel pseudouridine synthase-likegene containing the D18346 marker. Using the TblastN program thehigh-throughput human genomic sequence (HTGS) database (NCBI) wassearched to identify a PAC clone containing the human ortholog of thepseudouridine synthase-like gene. By repeated Blast searches of thehuman HTGS with portions of the sequence from this and overlapping PACand BAC clones we were able to form a contiguous map (“contig”) of 6overlapping BAC or PAC clones spanning approximately one million bp ofhuman genomic DNA sequence was found.

[0091] Using the Genscan gene prediction program we identified thepredicted exons and genes within this contig were identified. Twentythree genes were predicted in this region (FIG. 1A), including“pseudouridine synthase-like”, “cleavage and polyadenylation-like”, and“glycolipid transfer-like”; a few genes within this region had beenpreviously identified and/or experimentally verified by others (e.g.disheveled 1, dvl1). The Celera mouse genomic database was searched toidentify the murine orthologs of the genes within this region and piecedtogether the mouse contig (FIG. 1A).

Identification of a Novel Receptor, T1R3, within the Sac Region

[0092] In the screen of the million bp of genomic DNA sequence in theSac region, only one predicted GPCR gene was found. The gene, which wasreferred to as T1R3 (for taste receptor one, member three family ), wasof special interest because the predicted protein it encodes is mostsimilar to T1R1 and T1R2, two orphan GPCRs expressed in taste cells(17), and because, as will be shown below, it is expressed specificallyin taste cells. Human T1R3 (hT1R3) is located about 20 kb from thepseudouridine synthase-like gene, the human ortholog of the mouse genecontaining the D18346 marker (FIG. 1A). If T1R3 is Sac, then itsproximity to D18346 is consistent with the previously observed very lowprobability of crossovers between the marker and the Sac locus in F2crosses and congenic mice (16).

[0093] The intron/exon structure of the coding portion of the hT1R3 genewas predicted by Genscan to span 4 kb and contain 7 exons (FIG. 1B). Toconfirm and refine the inferred amino acid sequence of the predictedhT1R3 protein we cloned and sequenced multiple independent products frompolymerase chain reaction (PCR) amplified hT1R3 cDNAs derived from ahuman taste cDNA library. Based on the nucleotide sequence of thegenomic DNA and cDNAs, the hydrophobicity profile and TMpred predictionsof membrane spanning regions (FIG. 1C), hT1R3 is predicted to encode aprotein of 843 amino acids with seven transmembrane helices and a large558 amino acid long extracellular domain.

[0094] The corresponding mouse T1R3 (mT1R3) genomic sequence wasassembled from the Celera mouse genomic fragment database. Severalreverse transcriptase (RT)-PCR-generated mouse T1R3 cDNAs derived fromtaste bud mRNA of different mouse strains were also cloned andsequenced. The coding portion of the mouse T1R3 gene from C57BL/6 spans4 kb and contains 6 exons; the encoded protein is 858 amino acids long.Polymorphic differences between taster and non-taster strains of mice,and their potential functional significance, are described below (seeFIGS. 5 and 6 and related text).

[0095] T1R3 is a member of the family 3 subtype of GPCRs, all of whichcontain large extracellular domains. Other family 3 subtype GPCRsinclude metabotropic glutamate receptors (mGluR), extracellular calciumsensing receptors (ECaSR), candidate pheromone receptors expressed inthe vomeronasal organ (V2R), and two taste receptors, T1R1 and T1R2, ofunknown ligand specificity. T1R3 is most closely related to T1R1 andT1R2, sharing ˜30% amino acid sequence identity with each of theseorphan taste receptors (T1R1 and T1R2 are ˜40% identical to each other).At the amino acid level hT1R3 is ˜20% identical to mGluRs and ˜23%identical to ECaSRs. The large amino terminal domain (ATD) of family 3GPCRs has been implicated in ligand binding and dimerization (19). Likeother family 3 GPCRs, mT1R3 has an amino-terminal signal sequence, anextensive ATD of 573 amino acids, multiple predicted asparagine-linkedglycosylation sites (one of which is highly conserved), and severalconserved cysteine residues. Nine of these cysteines are within a regionthat links the ATD to the portion of the receptor containing thetransmembrane domains. The potential relevance of mT1R3's ATD inphenotypic differences between taster and non-taster strains of mice iselaborated below (see FIGS. 5 and 6 and related text).

Expression of T1R3 mRNA and Protein in Taste Tissue and Taste Buds

[0096] To examine the general distribution of mouse T1R3 in taste andnon-taste tissues, northern blot analysis was carried out with a panelof mouse mRNAs. The mouse T1R3 probe hybridized to a 7.2 kb mRNA presentin taste tissue, but not expressed in control lingual tissue devoid oftaste buds (non-taste) or in any of the several other tissues examined(FIG. 2A). A somewhat larger (˜7.8 kb) mRNA species was expressed atmoderate levels in testis, and at very low levels in brain. A smaller(˜6.7 kb) mRNA species was expressed at very low levels in thymus. The7.2 kb taste-expressed transcript is longer than the isolated cDNAs orGenscan predicted exons, suggesting that additional untranslatedsequences may be present in the transcript.

[0097] As another measure of the pattern of expression of T1R3 invarious tissues the expressed sequence tags (est) database were examinedfor strong matches to T1R3 and other predicted genes in the Sac region(FIG. 2B). While dvl1, glycolipid transfer-like, cleavage andpolyadenylation-like, and pseudouridine synthase-like genes each hadnumerous highly significant matches to ests from several differenttissues, T1R3 showed only a single strong match to an est from colon.This result, consistent with the northern, suggests that expression ofT1R3 is highly restricted—such a pattern of under-representation in theest database would fit with T1R3 being a taste receptor.

[0098] To determine the cellular pattern of T1R3 expression in tastetissue, in situ hybridization was performed: T1R3 was selectivelyexpressed in taste receptor cells, but absent from the surroundinglingual epithelium, muscle or connective tissue (FIG. 3A). Sense probecontrols showed no non-specific hybridization to lingual tissue (FIG.3A). The RNA hybridization signal for T1R3 was even stronger than thatfor α-gustducin (FIG. 3A), suggesting that T1R3 mRNA is very highlyexpressed in taste receptor cells. This is in contrast to results withT1R1 and T1R2 mRNAs, which are apparently expressed at lower levels thanis α-gustducin (17). Furthermore, T1R3 is highly expressed in taste budsfrom fungiform, foliate and circumvallate papillae, whereas T1R1 andT1R2 mRNAs each show different regionally variable patterns ofexpression (T1R1 is preferentially expressed in taste cells of thefungiform papillae and geschmacksstreifen (‘taste stripe’), to a lesserextent in those of the foliate papillae, but rarely in those of thecircumvallate papillae; T1R2 is commonly expressed in taste cells of thecircumvallate and foliate papillae, but rarely in those of the fungiformpapillae or geschmacksstreifen) (17).

[0099] To determine if T1R3 mRNA is expressed in particular subsets oftaste receptor cells, expression profiling was used (3). First, probesfrom the 3′regions of mouse clones for T1R3, α-gustducin, Gγ13, PLCβ2and G3PDH cDNAs were hybridized to RT-PCR-amplified cDNAs from a singlecircumvallate papilla vs. a similar-sized piece of non-gustatory lingualepithelium. In this way it was determined that mouse T1R3, likeα-gustducin, Gγ13 and PLCβ2, was expressed in taste bud-containingtissue, but not in non-gustatory lingual epithelia (FIG. 3B left). Thepattern of expression of these genes in individual taste cells was nextprofiled: the single cell RT-PCR products were hybridized with the sameset of probes used above. As previously determined (3), all of thenineteen α-gustducin-positive cells expressed Gβ3 and Gγ13; thesenineteen cells also all expressed PLCβ2 (FIG. 3B right). Twelve of thesenineteen cells (63%) also expressed T1R3. Only one of the five cellsthat were α-gustducin/Gβ3/Gγ13/PLCβ2-negative expressed T1R3. From thisit was concluded that expression of T1R3 and α-gustducin/Gβ3/Gγ13/PLCβ2,although not fully coincident, overlaps to a great extent. Thiscontrasts with previous in situ hybridization results with tastereceptor cells of the foliate papillae in which ˜15% ofα-gustducin-positive cells were positive for T1R1 or T1R2 (17).

[0100] Immunocytochemistry with an anti-hT1R3 antibody demonstrated thatabout one fifth of taste receptor cells in human circumvallate (FIG.4AC) and fungiform (FIG. 4EH) papillae were positive for hT1R3. hT1R3immunoreactivity was blocked by pre-incubation of the hT1R3 antibodywith the cognate peptide (FIG. 4B). Longitudinal sections of thehT1R3-postive taste cells displayed an elongated bipolar morphologytypical of so called light cells (many of which areα-gustducin-positive), with the immunoreactivity most prominent at ornear the taste pore (FIG. 4ACEH). Labeling adjacent sections withantibodies directed against hT1R3 and PLCβ2 showed more cells positivefor PLCβ2 than for hT1R3 (FIG. 4CD). Double labeling for hT1R3 and PLCβ2(FIG. 4EFG), or for hT1R3 and α-gustducin (FIG. 4HIJ) showed many, butnot all, cells to be doubly positive (more cells were positive for PLCβ2or α-gustducin than for hT1R3), consistent with the results fromexpression profiling. In sum, T1R3 mRNA and protein are selectivelyexpressed in a subset of “-gustducin/PLC$2- positive taste receptorcells as would be expected for a taste receptor.

A Single Polymorphic Difference in T1R3 may Explain the Sac^(d)Non-Taster Phenotype

[0101] C57BL/6 mice carrying the Sac^(b) allele and other so-calledtaster strains of mice display enhanced preferences and larger chordatympani nerve responses vs. DBA/2 mice (sac^(d)) and other non-tasterstrains for several compounds that humans characterize as sweet (e.g.sucrose, saccharin, acesulfame, dulcin and glycine) (10-12, 14, 15, 18).The inferred amino acid sequence of T1R3 from taster and non-tasterstrains of mice were examined looking for changes that might explainthese phenotypic differences (see FIG. 5A). All four non-taster strains(DBA/2, 129/Svev, BALB/c and C3H/HeJ) examined had identical nucleotidesequences despite the fact that their most recent common ancestors dateback to the early 1900s or earlier (see FIG. 5B). All four tasterstrains (C57BL/6J, SWR ,FVB/N and ST/bj) shared four nucleotidedifferences vs. the non-tasters: nt₁₃₅A→G, nt₁₆₃A→G, nt₁₇₉T→C andnt₆₅₂T→C (the taster nt is listed first). C57BL/6J also had a number ofpositions at which it differed from all other strains (see FIG. 5A),however, many of these differences were either “silent” alternate codonchanges in protein coding regions or substitutions within introns wherethey would be unlikely to have any pronounced effect. The two codingchanges (described as single letter amino acid changes at specificresidues; the taster aa is listed first) were T55A and I60T. The I60Tchange is a particularly intriguing difference as it is predicted tointroduce a novel N-linked glycosylation site in the ATD of T1R3 (seebelow)

[0102] To consider the functional relevance of these two amino aciddifferences in the T1R3 proteins from taster vs. non-taster, the ATD ofT1R3 was aligned with those of other members of the type 3 subset ofGPCRs (FIG. 6) and the ATD of T1R3 was modeled based on the recentlysolved structure of the ATD of the related mGluR1 receptor (19) (FIG.7). The ATD of T1R3 displays 28, 30, 24, and 20% identity to those ofT1R1, T1R2, CaSR and mGluR1, respectively (FIG. 6). 55 residues of ˜570in the ATD were identical among all five receptors. Included among theseconserved residues is a predicted N-linked glycosylation site at N85 ofT1R3. Based on homology to mGluR1, regions predicted to be involved indimerization of T1R3 are aa 55-60, 107-118, 152-160, and 178-181 (shownin FIG. 6 within dashed boxes) The I60T taster to non-tastersubstitution is predicted to introduce a novel N-linked glycosylationsite 27 amino acids upstream from the conserved N-linked glycosylationsite present in all five 5 receptors. The new N-linked glycosylationsite at N58 might interfere with normal glycosylation of the conservedsite at N85, alter the structure of the ligand binding domain, interferewith potential dimerization of the receptor, or have some other effecton T1R3 function.

[0103] To determine if glycosylation at N58 of the non-taster variant ofmT1R3 might be expected to alter the function of the protein we modeledits ATD on that of mGluR1 (19) (FIG. 7). The regions of potentialdimerization in T1R3 are very similar to those of mGluR1 and the aminoacids in these regions form tight fitting contact surfaces that suggestthat dimerization is indeed likely in T1R3. From the model of the threedimensional-structure of the ATD of T1R3 we can see that the novelN-linked glycosylation site at N58 would have a profound effect onT1R3's ability to dimerize (FIG. 7C). The addition of even a shortcarbohydrate group at N58 (a tri-saccharide moiety has been added in themodel in FIG. 7C) would disrupt at least one of the contact surfacesrequired for stability of the dimer. Therefore, if T1R3, like mGluR1,adopts a dimeric form (either homodimer or heterodimer), then thepredicted N-linked glycosyl group at N58 would be expected to precludeT1R3 from forming self-homodimers or heterodimers with any other GPCRsco-expressed with T1R3 using the same dimerization interface. Even ifthe novel predicted glycosylation site at N58 of non-taster T1R3 is notutilized, the T55A and I60T substitutions at the predicted surface ofdimerization may themselves affect the ability of T1R3 to form dimers.

REFERENCES

[0104] 1. McLaughlin, S. K. et al., 1992, Nature 357, 563-569

[0105] 2. Wong, G. T. et al., 1996, Nature 381, 796-800

[0106] 3. Huang, L. et al., 1999, Nat Neurosci 2, 1055-1062

[0107] 4. Adler, E. et al., 2000. Cell 10, 693-702

[0108] 5. Chandrashekar, J. et al., 2000, Cell 100, 703-711

[0109] 6. Matsunami H et al. Nature 2000, 404:601-604

[0110] 7. Lindemann, B. 1996, Physiol. Rev. 76, 719-766

[0111] 8. Gilbertson, T. A. et al., 2000, Curr. Olin. Neurobiol. 10,519-527

[0112] 9. Fuller, J. L. 1974, J Hered 65, 33-36.

[0113] 10. Lush, I. E. 1989 Genet. Res. 53, 95-99

[0114] 11. Capeless, C. G. and Whitney, G. 1995, Chem Senses 20, 291-298

[0115] 12. Lush, I. E. et al., 1995, Genet Res 66, 167-174

[0116] 13. Ninomiya Y. et al. In Chemical Senses Vol. 3, Genetics ofPerception and Communication (ed. C. J. Wysocki and M. R. Kare). NewYork: Marcel Dekker. pp 267-278 (1991).

[0117] 14. Bachmanov, A. A. et al., 1977, Mammal Genome 8, 545-548

[0118] 15. Blizzard, D. A. et al, 1999, Chem Senses 24, 373-385

[0119] 16. Li, X. et al, 2001, Genome 12:13-16

[0120] 17. Hoon, M. A. et al., 1999, Cell 96, 541-551

[0121] 18. Frank, M. E. and Blizard, D. A. 1999, Physiol Behav. 67,287-297

[0122] 19. Kunishima, N. et al., 2000, Nature 407, 971-977

[0123] 20. Capretta, P. J., 1970, Psychogenomic Science 21, 133-135

[0124] 21. Pelz, W. et al., 1973, Physiology and Behavior 10, 263-265

[0125] 22. Lush, I. E., 1984, Genet. Res. 44, 151-160

[0126] 23. Lush, I. E., 1986, Genet. Res. 47, 117-123

[0127] 24. Lush, I. E. and Holland, G., 1988, Genet. Res. 52, 207-212

[0128] 25. Capeless, C. G. et al., 1992, Behav. Genet. 22, 655-663

[0129] 26. Kinnamon, S. C. and Margolskee, R. F., 1996, Current Opinionin Neurobiology. 6, 506-513

[0130] 27. Lum, C. K. L., and Cagan, R. H., 1976, Biochim. Biophys. Acta421, 380-394.

[0131] 28. Cagan, R. H., and Morris, R. W., 1979, Proc. Natl. Acad. Sci.USA 76, 1692-1696

[0132] 29. Farbman, A. I. et al., 1987, Scan. Microsc. 1, 351-357

[0133] 30. Shimazaki, K. et al.,1986, Biochim. Biophys. Acta 884,291-298

[0134] 31. Hiji, T., 1975, Nature 256, 427-429

[0135] 32. Bernhardt, S. J. et al., J Physiol, 490, 325-336.

[0136] 33. Nakashima, K., and Ninomiya, Y., 1998, Cell Physiol Biochem8, 224-230

[0137] 34. Ming, D. et al., 1998, Proc Natl Acad Sci USA 95, 8933-8938

[0138] 35. Imoto, T. et al., 1991, Comp. Biochem. Physiol. A Physiol.100, 309-314

[0139] 36. Ninomiya Y. et al., 1995, Am. J. Physiol. 268, R1019-R1025

[0140] 37. Ninomiya Y. et al., 1999, J. Neurophysiol. 81, 3087-3091

[0141] 38. Rossler, P. et al. 1998, Eur J Cell Biol 77, 253-61

[0142] 39. Smith, D. V., Margolis, F. L., 1999, Curr Biol 9, R453-455

[0143] 40. Ninomiya, Y. et al., 1997, Am J Physiol 272, R1002-R1006

[0144] 41. Yamaguchi, S., 1991, Physiol. Behav. 49, 833-841

[0145] 42. Chaudhari, N., and Roper, S. D., 1998, Ann. NY Acad. Sci.855, 398-406

[0146] 43. Chaudhari, N. et al., 2000, Nat. Neurosci. 3, 113-119

[0147] 44. Danilova, V. et al., 1999, Sus scrofa. Chem Senses 24,301-316

[0148] 45. Ninomiya, Y. et al., 2000, J Nutr. 130, 950S-953S

[0149] 46. Bakre, M. M. et al., 2001, Submitted (2001).

[0150] 47. Hogan, B., Beddington, R., Costantini, F. & Lacy, E.Manipulating the mouse embryo: a laboratory manual, (Cold Spring HarborLaboratory, Cold Spring Harbor, 1994).

[0151] 48. Thompson, J. D. et al., Nucleic Acids Res. 22, pp. 4673-4680.

[0152] 49. Sali, A. and Blundell, T. L., 1993, J Mol. Biol 234, 779-815.

[0153] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description andaccompanying figures. Such modifications are intended to fall within thescope of the appended claims. Various references are cited herein, thedisclosures of which are incorporated by reference in their entireties.

1 12 1 343 DNA Homo sapiens CDS (151)..(342) 1 ggacaccact ggggccccagggtgtggcaa gtgaggatgg caagggtttt gctaaacaaa 60 tcctctgccc gctccccgccccgggctcac tccatgtgag gccccagtcg gggcagccac 120 ctgccgtgcc tgttggaagttgcctctgcc atg ctg ggc cct gct gtc ctg ggc 174 Met Leu Gly Pro Ala ValLeu Gly 1 5 ctc agc ctc tgg gct ctc ctg cac cct ggg acg ggg gcc cca ttgtgc 222 Leu Ser Leu Trp Ala Leu Leu His Pro Gly Thr Gly Ala Pro Leu Cys10 15 20 ctg tca cag caa ctt agg atg aag ggg gac tac gtg ctg ggg ggg ctg270 Leu Ser Gln Gln Leu Arg Met Lys Gly Asp Tyr Val Leu Gly Gly Leu 2530 35 40 ttc ccc ctg ggc gag gcc gag gag gct ggc ctc cgc agc cgg aca cgg318 Phe Pro Leu Gly Glu Ala Glu Glu Ala Gly Leu Arg Ser Arg Thr Arg 4550 55 ccc agc agc cct gtg tgc acc agg t 343 Pro Ser Ser Pro Val Cys ThrArg 60 2 305 DNA Homo sapiens CDS (4)..(303) 2 agg ttc tcc tca aac ggcctg ctc tgg gca ctg gcc atg aaa atg gcc 48 Phe Ser Ser Asn Gly Leu LeuTrp Ala Leu Ala Met Lys Met Ala 1 5 10 15 gtg gag gag atc aac aac aagtcg gat ctg ctg ccc ggg ctg cgc ctg 96 Val Glu Glu Ile Asn Asn Lys SerAsp Leu Leu Pro Gly Leu Arg Leu 20 25 30 ggc tac gac ctc ttt gat acg tgctcg gag cct gtg gtg gcc atg aag 144 Gly Tyr Asp Leu Phe Asp Thr Cys SerGlu Pro Val Val Ala Met Lys 35 40 45 ccc agc ctc atg ttc ctg gcc aag gcaggc agc cgc gac atc gcc gcc 192 Pro Ser Leu Met Phe Leu Ala Lys Ala GlySer Arg Asp Ile Ala Ala 50 55 60 tac tgc aac tac acg cag tac cag ccc cgtgtg ctg gct gtc atc ggg 240 Tyr Cys Asn Tyr Thr Gln Tyr Gln Pro Arg ValLeu Ala Val Ile Gly 65 70 75 ccc cac tcg tca gag ctc gcc atg gtc acc ggcaag ttc ttc agc ttc 288 Pro His Ser Ser Glu Leu Ala Met Val Thr Gly LysPhe Phe Ser Phe 80 85 90 95 ttc ctc atg ccc cag gt 305 Phe Leu Met ProGln 100 3 787 DNA Homo sapiens CDS (3)..(785) 3 ag gtc agc tac ggt gctagc atg gag ctg ctg agc gcc cgg gag acc 47 Val Ser Tyr Gly Ala Ser MetGlu Leu Leu Ser Ala Arg Glu Thr 1 5 10 15 ttc ccc tcc ttc ttc cgc accgtg ccc agc gac cgt gtg cag ctg acg 95 Phe Pro Ser Phe Phe Arg Thr ValPro Ser Asp Arg Val Gln Leu Thr 20 25 30 gcc gcc gcg gag ctg ctg cag gagttc ggc tgg aac tgg gtg gcc gcc 143 Ala Ala Ala Glu Leu Leu Gln Glu PheGly Trp Asn Trp Val Ala Ala 35 40 45 ctg ggc agc gac gac gag tac ggc cggcag ggc ctg agc atc ttc tcg 191 Leu Gly Ser Asp Asp Glu Tyr Gly Arg GlnGly Leu Ser Ile Phe Ser 50 55 60 gcc ctg gcc gcg gca cgc ggc atc tgc atcgcg cac gag ggc ctg gtg 239 Ala Leu Ala Ala Ala Arg Gly Ile Cys Ile AlaHis Glu Gly Leu Val 65 70 75 ccg ctg ccc cgt gcc gat gac tcg cgg ctg gggaag gtg cag gac gtc 287 Pro Leu Pro Arg Ala Asp Asp Ser Arg Leu Gly LysVal Gln Asp Val 80 85 90 95 ctg cac cag gtg aac cag agc agc gtg cag gtggtg ctg ctg ttc gcc 335 Leu His Gln Val Asn Gln Ser Ser Val Gln Val ValLeu Leu Phe Ala 100 105 110 tcc gtg cac gcc gcc cac gcc ctc ttc aac tacagc atc agc agc agg 383 Ser Val His Ala Ala His Ala Leu Phe Asn Tyr SerIle Ser Ser Arg 115 120 125 ctc tcg ccc aag gtg tgg gtg gcc agc gag gcctgg ctg acc tct gac 431 Leu Ser Pro Lys Val Trp Val Ala Ser Glu Ala TrpLeu Thr Ser Asp 130 135 140 ctg gtc atg ggg ctg ccc ggc atg gcc cag atgggc acg gtg ctt ggc 479 Leu Val Met Gly Leu Pro Gly Met Ala Gln Met GlyThr Val Leu Gly 145 150 155 ttc ctc cag agg ggt gcc cag ctg cac gag ttcccc cag tac gtg aag 527 Phe Leu Gln Arg Gly Ala Gln Leu His Glu Phe ProGln Tyr Val Lys 160 165 170 175 acg cac ctg gcc ctg gcc acc gac ccg gccttc tgc tct gcc ctg ggc 575 Thr His Leu Ala Leu Ala Thr Asp Pro Ala PheCys Ser Ala Leu Gly 180 185 190 gag agg gag cag ggt ctg gag gag gac gtggtg ggc cag cgc tgc ccg 623 Glu Arg Glu Gln Gly Leu Glu Glu Asp Val ValGly Gln Arg Cys Pro 195 200 205 cag tgt gac tgc atc acg ctg cag aac gtgagc gca ggg cta aat cac 671 Gln Cys Asp Cys Ile Thr Leu Gln Asn Val SerAla Gly Leu Asn His 210 215 220 cac cag acg ttc tct gtc tac gca gct gtgtat agc gtg gcc cag gcc 719 His Gln Thr Phe Ser Val Tyr Ala Ala Val TyrSer Val Ala Gln Ala 225 230 235 ctg cac aac act ctt cag tgc aac gcc tcaggc tgc ccc gcg cag gac 767 Leu His Asn Thr Leu Gln Cys Asn Ala Ser GlyCys Pro Ala Gln Asp 240 245 250 255 ccc gtg aag ccc tgg cag gt 787 ProVal Lys Pro Trp Gln 260 4 208 DNA Homo sapiens CDS (3)..(206) 4 ag ctcctg gag aac atg tac aac ctg acc ttc cac gtg ggc ggg ctg 47 Leu Leu GluAsn Met Tyr Asn Leu Thr Phe His Val Gly Gly Leu 1 5 10 15 ccg ctg cggttc gac agc agc gga aac gtg gac atg gag tac gac ctg 95 Pro Leu Arg PheAsp Ser Ser Gly Asn Val Asp Met Glu Tyr Asp Leu 20 25 30 aag ctg tgg gtgtgg cag ggc tca gtg ccc agg ctc cac gac gtg ggc 143 Lys Leu Trp Val TrpGln Gly Ser Val Pro Arg Leu His Asp Val Gly 35 40 45 agg ttc aac ggc agcctc agg aca gag cgc ctg aag atc cgc tgg cac 191 Arg Phe Asn Gly Ser LeuArg Thr Glu Arg Leu Lys Ile Arg Trp His 50 55 60 acg tct gac aac cag gt208 Thr Ser Asp Asn Gln 65 5 125 DNA Homo sapiens CDS (3)..(122) 5 agaag ccc gtg tcc cgg tgc tcg cgg cag tgc cag gag ggc cag gtg 47 Lys ProVal Ser Arg Cys Ser Arg Gln Cys Gln Glu Gly Gln Val 1 5 10 15 cgc cgggtc aag ggg ttc cac tcc tgc tgc tac gac tgt gtg gac tgc 95 Arg Arg ValLys Gly Phe His Ser Cys Cys Tyr Asp Cys Val Asp Cys 20 25 30 gag gcg ggcagc tac cgg caa aac cca ggt 125 Glu Ala Gly Ser Tyr Arg Gln Asn Pro 3540 6 961 DNA Homo sapiens CDS (2)..(958) 6 a gac gac atc gcc tgc acc ttttgt ggc cag gat gag tgg tcc ccg gag 49 Asp Asp Ile Ala Cys Thr Phe CysGly Gln Asp Glu Trp Ser Pro Glu 1 5 10 15 cga agc aca cgc tgc ttc cgccgc agg tct cgg ttc ctg gca tgg ggc 97 Arg Ser Thr Arg Cys Phe Arg ArgArg Ser Arg Phe Leu Ala Trp Gly 20 25 30 gag ccg gct gtg ctg ctg ctg ctcctg ctg ctg agc ctg gcg ctg ggc 145 Glu Pro Ala Val Leu Leu Leu Leu LeuLeu Leu Ser Leu Ala Leu Gly 35 40 45 ctt gtg ctg gct gct ttg ggg ctg ttcgtt cac cat cgg gac agc cca 193 Leu Val Leu Ala Ala Leu Gly Leu Phe ValHis His Arg Asp Ser Pro 50 55 60 ctg gtt cag gcc tcg ggg ggg ccc ctg gcctgc ttt ggc ctg gtg tgc 241 Leu Val Gln Ala Ser Gly Gly Pro Leu Ala CysPhe Gly Leu Val Cys 65 70 75 80 ctg ggc ctg gtc tgc ctc agc gtc ctc ctgttc cct ggc cag ccc agc 289 Leu Gly Leu Val Cys Leu Ser Val Leu Leu PhePro Gly Gln Pro Ser 85 90 95 cct gcc cga tgc ctg gcc cag cag ccc ttg tcccac ctc ccg ctc acg 337 Pro Ala Arg Cys Leu Ala Gln Gln Pro Leu Ser HisLeu Pro Leu Thr 100 105 110 ggc tgc ctg agc aca ctc ttc ctg cag gcg gccgag atc ttc gtg gag 385 Gly Cys Leu Ser Thr Leu Phe Leu Gln Ala Ala GluIle Phe Val Glu 115 120 125 tca gaa ctg cct ctg agc tgg gca gac cgg ctgagt ggc tgc ctg cgg 433 Ser Glu Leu Pro Leu Ser Trp Ala Asp Arg Leu SerGly Cys Leu Arg 130 135 140 ggg ccc tgg gcc tgg ctg gtg gtg ctg ctg gccatg ctg gtg gag gtc 481 Gly Pro Trp Ala Trp Leu Val Val Leu Leu Ala MetLeu Val Glu Val 145 150 155 160 gca ctg tgc acc tgg tac ctg gtg gcc ttcccg ccg gag gtg gtg acg 529 Ala Leu Cys Thr Trp Tyr Leu Val Ala Phe ProPro Glu Val Val Thr 165 170 175 gac tgg cac atg ctg ccc acg gag gcg ctggtg cac tgc cgc aca cgc 577 Asp Trp His Met Leu Pro Thr Glu Ala Leu ValHis Cys Arg Thr Arg 180 185 190 tcc tgg gtc agc ttc ggc cta gcg cac gccacc aat gcc acg ctg gcc 625 Ser Trp Val Ser Phe Gly Leu Ala His Ala ThrAsn Ala Thr Leu Ala 195 200 205 ttt ctc tgc ttc ctg ggc act ttc ctg gtgcgg agc cag ccg ggc cgc 673 Phe Leu Cys Phe Leu Gly Thr Phe Leu Val ArgSer Gln Pro Gly Arg 210 215 220 tac aac cgt gcc cgt ggc ctc acc ttt gccatg ctg gcc tac ttc atc 721 Tyr Asn Arg Ala Arg Gly Leu Thr Phe Ala MetLeu Ala Tyr Phe Ile 225 230 235 240 acc tgg gtc tcc ttt gtg ccc ctc ctggcc aat gtg cag gtg gtc ctc 769 Thr Trp Val Ser Phe Val Pro Leu Leu AlaAsn Val Gln Val Val Leu 245 250 255 agg ccc gcc gtg cag atg ggc gcc ctcctg ctc tgt gtc ctg ggc atc 817 Arg Pro Ala Val Gln Met Gly Ala Leu LeuLeu Cys Val Leu Gly Ile 260 265 270 ctg gct gcc ttc cac ctg ccc agg tgttac ctg ctc atg cgg cag cca 865 Leu Ala Ala Phe His Leu Pro Arg Cys TyrLeu Leu Met Arg Gln Pro 275 280 285 ggg ctc aac acc ccc gag ttc ttc ctggga ggg ggc cct ggg gat gcc 913 Gly Leu Asn Thr Pro Glu Phe Phe Leu GlyGly Gly Pro Gly Asp Ala 290 295 300 caa ggc cag aat gac ggg aac aca ggaaat cag ggg aaa cat gag tga 961 Gln Gly Gln Asn Asp Gly Asn Thr Gly AsnGln Gly Lys His Glu 305 310 315 7 852 PRT Homo sapiens 7 Met Leu Gly ProAla Val Leu Gly Leu Ser Leu Trp Ala Leu Leu His 1 5 10 15 Pro Gly ThrGly Ala Pro Leu Cys Leu Ser Gln Gln Leu Arg Met Lys 20 25 30 Gly Asp TyrVal Leu Gly Gly Leu Phe Pro Leu Gly Glu Ala Glu Glu 35 40 45 Ala Gly LeuArg Ser Arg Thr Arg Pro Ser Ser Pro Val Cys Thr Arg 50 55 60 Phe Ser SerAsn Gly Leu Leu Trp Ala Leu Ala Met Lys Met Ala Val 65 70 75 80 Glu GluIle Asn Asn Lys Ser Asp Leu Leu Pro Gly Leu Arg Leu Gly 85 90 95 Tyr AspLeu Phe Asp Thr Cys Ser Glu Pro Val Val Ala Met Lys Pro 100 105 110 SerLeu Met Phe Leu Ala Lys Ala Gly Ser Arg Asp Ile Ala Ala Tyr 115 120 125Cys Asn Tyr Thr Gln Tyr Gln Pro Arg Val Leu Ala Val Ile Gly Pro 130 135140 His Ser Ser Glu Leu Ala Met Val Thr Gly Lys Phe Phe Ser Phe Phe 145150 155 160 Leu Met Pro Gln Val Ser Tyr Gly Ala Ser Met Glu Leu Leu SerAla 165 170 175 Arg Glu Thr Phe Pro Ser Phe Phe Arg Thr Val Pro Ser AspArg Val 180 185 190 Gln Leu Thr Ala Ala Ala Glu Leu Ser Gln Glu Phe GlyTrp Asn Trp 195 200 205 Val Ala Ala Leu Gly Ser Asp Asp Glu Tyr Gly ArgGln Gly Leu Ser 210 215 220 Ile Phe Ser Ala Leu Ala Ala Ala Arg Gly IleCys Ile Ala His Glu 225 230 235 240 Gly Leu Val Pro Leu Pro Arg Ala AspAsp Ser Arg Leu Gly Lys Val 245 250 255 Gln Asp Val Leu His Gln Val AsnGln Ser Ser Val Gln Val Val Leu 260 265 270 Leu Phe Ala Ser Val His AlaAla His Ala Leu Phe Asn Tyr Ser Ile 275 280 285 Ser Ser Arg Leu Ser ProLys Val Trp Val Ala Ser Glu Ala Trp Leu 290 295 300 Thr Ser Asp Leu ValMet Gly Leu Pro Gly Met Ala Gln Met Gly Thr 305 310 315 320 Val Leu GlyPhe Leu Gln Arg Gly Ala Gln Leu His Glu Phe Pro Gln 325 330 335 Tyr ValLys Thr His Leu Ala Leu Ala Thr Asp Pro Ala Phe Cys Ser 340 345 350 AlaLeu Gly Glu Arg Glu Gln Gly Leu Glu Glu Asp Val Val Gly Gln 355 360 365Arg Cys Pro Gln Cys Asp Cys Ile Thr Leu Gln Asn Val Ser Ala Gly 370 375380 Leu Asn His His Gln Thr Phe Ser Val Tyr Ala Ala Val Tyr Ser Val 385390 395 400 Ala Gln Ala Leu His Asn Thr Leu Gln Cys Asn Ala Ser Gly CysPro 405 410 415 Ala Gln Asp Pro Val Lys Pro Trp Gln Leu Leu Glu Asn MetTyr Asn 420 425 430 Leu Thr Phe His Val Gly Gly Leu Pro Leu Arg Phe AspSer Ser Gly 435 440 445 Asn Val Asp Met Glu Tyr Asp Leu Lys Leu Trp ValTrp Gln Gly Ser 450 455 460 Val Pro Arg Leu His Asp Val Gly Arg Phe AsnGly Ser Leu Arg Thr 465 470 475 480 Glu Arg Leu Lys Ile Arg Trp His ThrSer Asp Asn Gln Lys Pro Val 485 490 495 Ser Arg Cys Ser Arg Gln Cys GlnGlu Gly Gln Val Arg Arg Val Lys 500 505 510 Gly Phe His Ser Cys Cys TyrAsp Cys Val Asp Cys Glu Ala Gly Ser 515 520 525 Tyr Arg Gln Asn Pro AspAsp Ile Ala Cys Thr Phe Cys Gly Gln Asp 530 535 540 Glu Trp Ser Pro GluArg Ser Thr Arg Cys Phe Arg Arg Arg Ser Arg 545 550 555 560 Phe Leu AlaTrp Gly Glu Pro Ala Val Leu Leu Leu Leu Leu Leu Leu 565 570 575 Ser LeuAla Leu Gly Leu Val Leu Ala Ala Leu Gly Leu Phe Val His 580 585 590 HisArg Asp Ser Pro Leu Val Gln Ala Ser Gly Gly Pro Leu Ala Cys 595 600 605Phe Gly Leu Val Cys Leu Gly Leu Val Cys Leu Ser Val Leu Leu Phe 610 615620 Pro Gly Gln Pro Ser Pro Ala Arg Cys Leu Ala Gln Gln Pro Leu Ser 625630 635 640 His Leu Pro Leu Thr Gly Cys Leu Ser Thr Leu Phe Leu Gln AlaAla 645 650 655 Glu Ile Phe Val Glu Ser Glu Leu Pro Leu Ser Trp Ala AspArg Leu 660 665 670 Ser Gly Cys Leu Arg Gly Pro Trp Ala Trp Leu Val ValLeu Leu Ala 675 680 685 Met Leu Val Glu Val Ala Leu Cys Thr Trp Tyr LeuVal Ala Phe Pro 690 695 700 Pro Glu Val Val Thr Asp Trp His Met Leu ProThr Glu Ala Leu Val 705 710 715 720 His Cys Arg Thr Arg Ser Trp Val SerPhe Gly Leu Ala His Ala Thr 725 730 735 Asn Ala Thr Leu Ala Phe Leu CysPhe Leu Gly Thr Phe Leu Val Arg 740 745 750 Ser Gln Pro Gly Arg Tyr AsnArg Ala Arg Gly Leu Thr Phe Ala Met 755 760 765 Leu Ala Tyr Phe Ile ThrTrp Val Ser Phe Val Pro Leu Leu Ala Asn 770 775 780 Val Gln Val Val LeuArg Pro Ala Val Gln Met Gly Ala Leu Leu Leu 785 790 795 800 Cys Val LeuGly Ile Leu Ala Ala Phe His Leu Pro Arg Cys Tyr Leu 805 810 815 Leu MetArg Gln Pro Gly Leu Asn Thr Pro Glu Phe Phe Leu Gly Gly 820 825 830 GlyPro Gly Asp Ala Gln Gly Gln Asn Asp Gly Asn Thr Gly Asn Gln 835 840 845Gly Lys His Glu 850 8 490 PRT Mus musculus 8 Met Pro Ala Leu Ala Ile MetGly Leu Ser Leu Ala Ala Phe Leu Glu 1 5 10 15 Leu Gly Met Gly Ala SerLeu Cys Leu Ser Gln Gln Phe Lys Ala Gln 20 25 30 Tyr Ile Leu Gly Gly ProLeu Gly Ser Thr Glu Glu Ala Thr Leu Asn 35 40 45 Gln Arg Thr Gln Pro AsnSer Ile Pro Asn Arg Phe Ser Pro Leu Leu 50 55 60 Phe Leu Ala Met Lys MetAla Val Glu Glu Asn Gly Ser Ala Gly Leu 65 70 75 80 Arg Tyr Asp Leu PheThr Ser Glu Pro Val Val Thr Met Lys Ser Ser 85 90 95 Leu Met Phe Leu AlaLys Val Gly Ser Gln Ser Ile Ala Ala Tyr Cys 100 105 110 Asn Tyr Thr GlnTyr Gln Pro Arg Val Leu Ala Val Ile Gly Pro His 115 120 125 Ser Ser GluLeu Ala Leu Ile Thr Gly Lys Phe Phe Ser Phe Leu Met 130 135 140 Gln ValSer Ser Ala Ser Met Asp Arg Ser Asp Arg Glu Thr Phe Pro 145 150 155 160Ser Phe Phe Thr Val Ser Asp Arg Val Gln Leu Gln Ala Val Val Thr 165 170175 Leu Leu Gln Asn Phe Ser Asn Trp Val Ala Ala Leu Gly Ser Asp Asp 180185 190 Asp Arg Glu Gly Leu Ser Ile Phe Ser Ser Leu Ala Asn Ala Arg Gly195 200 205 Ile Ile Ala His Glu Gly Leu Val Pro Gln His Asp Thr Ser GlyGln 210 215 220 Gln Leu Gly Lys Val Leu Asp Val Leu Arg Gln Val Asn GlnSer Lys 225 230 235 240 Val Gln Val Val Leu Ala Ser Ala Arg Ala Val TyrSer Leu Phe Ser 245 250 255 Tyr Ser Ile His His Gly Leu Ser Pro Lys ValTrp Val Ala Glu Ser 260 265 270 Leu Thr Ser Asp Leu Val Met Thr Leu ProAsn Ile Ala Arg Val Thr 275 280 285 Val Leu Gly Phe Leu Gln Arg Gly AlaLeu Leu Pro Glu Phe Ser His 290 295 300 Tyr Val Glu Thr His Leu Ala LeuAla Ala Asp Pro Ala Phe Ala Ser 305 310 315 320 Leu Asn Ala Glu Leu AspLeu Glu Glu His Val Met Gly Gln Arg Cys 325 330 335 Pro Arg Asp Asp IleMet Leu Gln Asn Leu Ser Ser Gly Leu Leu Gln 340 345 350 Asn Leu Ser AlaGly Gln Leu His His Gln Ile Phe Ala Thr Tyr Ala 355 360 365 Val Tyr SerVal Gln Ala His Asn Thr Leu Gln Asn Val Ser His His 370 375 380 Val SerGlu His Val Leu Pro Trp Gln Leu Glu Asn Met Tyr Asn Met 385 390 395 400Ser His Ala Arg Asp Leu Thr Leu Gln Ala Glu Asn Val Asp Met Glu 405 410415 Tyr Asp Leu Lys Met Trp Val Trp Gln Ser Pro Thr Pro Val Leu His 420425 430 Thr Val Gly Thr Phe Asn Gly Thr Gln Leu Gln Gln Ser Lys Met Tyr435 440 445 Trp Pro Gly Asn Gln Pro Val Gln Ser Arg Gln Lys Asp Gln ValArg 450 455 460 Arg Val Lys Gly Phe His Ser Tyr Asp Val Asp Lys Ala GlySer Tyr 465 470 475 480 Arg Lys His Pro Asp Asp Phe Thr Thr Pro 485 4909 480 PRT Rattus norvegicus 9 Met Leu Phe Trp Ala Ala His Leu Leu LeuSer Leu Gln Leu Val Tyr 1 5 10 15 Cys Trp Ala Phe Ser Cys Gln Arg ThrGlu Ser Ser Pro Gly Phe Ser 20 25 30 Leu Pro Phe Leu Leu Ala Gly Ser LeuHis Gly Asp Cys Leu Gln Val 35 40 45 Arg His Arg Pro Leu Val Thr Ser AspArg Pro Asp Ser Phe Asn Gly 50 55 60 His Tyr His Leu Phe Gln Arg Phe ThrVal Glu Glu Asn Ser Ser Ala 65 70 75 80 Asn Ile Thr Tyr Glu Leu Tyr ValSer Glu Ser Ala Asn Val Tyr Ala 85 90 95 Thr Leu Arg Val Leu Ala Leu GlnGly Pro Arg His Ile Glu Ile Gln 100 105 110 Lys Asp Leu Arg Asn His SerSer Lys Val Val Ala Phe Ile Pro Asp 115 120 125 Asn Thr Asp His Ala ValThr Thr Ala Ala Leu Leu Gly Pro Leu Met 130 135 140 Leu Val Ser Glu AlaSer Ser Val Val Ser Ala Lys Arg Lys Phe Pro 145 150 155 160 Ser Phe LeuThr Val Ser Asp Arg His Gln Val Glu Val Met Val Gln 165 170 175 Leu LeuGln Ser Phe Gly Val Trp Ile Ser Leu Ile Gly Ser Asp Tyr 180 185 190 GlyGln Leu Gly Val Gln Ala Leu Glu Glu Leu Ala Val Pro Arg Gly 195 200 205Ile Val Ala Phe Lys Asp Ile Val Pro Phe Ser Ala Arg Val Gly Asp 210 215220 Pro Arg Met Gln Ser Met Met Gln His Leu Ala Gln Ala Arg Thr Thr 225230 235 240 Val Val Val Ser Asn Arg His Leu Ala Arg Val Phe Phe Arg SerVal 245 250 255 Val Leu Ala Asn Leu Thr Gly Lys Val Trp Val Ala Glu AspAla Ile 260 265 270 Ser Thr Tyr Ile Thr Ser Val Thr Gly Ile Gln Gly IleThr Val Leu 275 280 285 Gly Val Ala Val Gln Gln Arg Gln Val Pro Gly LeuLys Glu Phe Glu 290 295 300 Glu Ser Tyr Val Arg Ala Val Thr Ala Ala ProSer Ala Pro Glu Gly 305 310 315 320 Ser Trp Ser Thr Cys Asn Gln Leu ArgGlu Cys His Thr Phe Thr Thr 325 330 335 Arg Asn Met Pro Thr Leu Gly AlaPhe Ser Met Ser Ala Ala Tyr Arg 340 345 350 Val Tyr Glu Val Ala Val HisGly His Gln Leu Leu Gly Thr Ser Glu 355 360 365 Ile Ser Arg Gly Pro ValTyr Pro Trp Gln Leu Gln Gln Ile Tyr Lys 370 375 380 Val Asn Leu Leu HisGlu Asn Thr Val Ala Asp Asn Asp Thr Leu Gly 385 390 395 400 Tyr Tyr AspIle Ile Ala Trp Asp Trp Asn Gly Pro Glu Trp Thr Phe 405 410 415 Glu IleIle Gly Ser Ala Ser Leu Ser Pro Val His Asp Ile Asn Lys 420 425 430 ThrLys Ile Gln Trp His Gly Lys Asn Asn Gln Pro Val Val Thr Thr 435 440 445Asp Leu Ala His His Arg Val Val Val Gly Ser His His Phe Glu Val 450 455460 Pro Glu Ala Gly Thr Phe Leu Asn Met Ser Glu Leu His Ile Gln Pro 465470 475 480 10 484 PRT Rattus norvegicus 10 Met Gly Pro Gln Ala Arg ThrLeu Cys Leu Leu Ser Leu Leu Leu His 1 5 10 15 Val Leu Pro Lys Pro GlyLys Leu Val Glu Asn Ser Asp Phe His Leu 20 25 30 Ala Tyr Leu Leu Gly GlyThr Leu His Ala Asn Val Lys Ser Ile Ser 35 40 45 His Leu Ser Tyr Leu GlnVal Pro Lys Asn Glu Phe Thr Met Lys Val 50 55 60 Leu Tyr Asn Leu Met GlnArg Phe Ala Val Glu Glu Asn Cys Ser Ser 65 70 75 80 Gly Val Leu Tyr GluMet Val Val Tyr Leu Ser Asn Asn Ile His Pro 85 90 95 Gly Leu Tyr Phe LeuAla Gln Asp Asp Asp Leu Leu Pro Ile Leu Lys 100 105 110 Asp Tyr Ser GlnTyr Met Pro His Val Val Ala Val Ile Pro Asp Asn 115 120 125 Ser Glu SerAla Ile Thr Val Ser Asn Ile Leu Ser His Leu Ile Gln 130 135 140 Ile ThrSer Ala Ile Ser Asp Lys Arg Asp Lys Arg His Phe Pro Ser 145 150 155 160Met Leu Thr Val Ser Ala Thr His His Ile Glu Ala Met Val Gln Leu 165 170175 Met Val His Phe Gln Asn Trp Ile Val Val Leu Val Ser Asp Asp Asp 180185 190 Arg Glu Asn Ser His Leu Leu Ser Gln Arg Leu Thr Lys Thr Ser Asp195 200 205 Ile Ile Ala Phe Gln Glu Val Leu Pro Ile Pro Glu Ser Ser GlnVal 210 215 220 Met Arg Ser Glu Glu Gln Arg Gln Leu Asp Asn Ile Leu AspLys Leu 225 230 235 240 Arg Arg Thr Ser Ala Arg Val Val Val Ser Pro GluLeu Ser Leu Tyr 245 250 255 Ser Phe Phe His Glu Val Leu Arg Trp Asn PheThr Gly Phe Val Trp 260 265 270 Ile Ala Glu Ser Ala Ile Asp Pro Val LeuHis Asn Leu Thr Glu Leu 275 280 285 Arg His Thr Thr Phe Leu Gly Val ThrIle Gln Arg Val Ser Ile Pro 290 295 300 Gly Phe Ser Gln Phe Arg Val ArgArg Asp Lys Pro Gly Tyr Pro Val 305 310 315 320 Pro Asn Thr Thr Asn LeuArg Thr Thr Asn Gln Asp Asp Ala Cys Leu 325 330 335 Asn Thr Thr Lys SerPhe Asn Asn Ile Leu Ile Leu Ser Gly Glu Arg 340 345 350 Val Val Tyr SerVal Tyr Ser Val Ala Val His Ala His Arg Leu Leu 355 360 365 Gly Asn ArgVal Arg Thr Lys Gln Lys Val Tyr Pro Trp Gln Leu Arg 370 375 380 Glu IleTrp His Val Asn Thr Leu Leu Gly Asn Arg Leu Phe Gln Gln 385 390 395 400Asp Met Pro Met Leu Leu Asp Ile Ile Gln Trp Gln Trp Asp Leu Ser 405 410415 Gln Asn Pro Phe Gln Ser Ile Ala Ser Tyr Ser Pro Thr Ser Lys Arg 420425 430 Thr Tyr Ile Asn Asn Val Ser Trp Tyr Thr Pro Asn Asn Thr Pro Val435 440 445 Met Ser Lys Ser Gln Pro Gln Met Lys Lys Ser Val Gly Leu HisPro 450 455 460 Phe Glu Leu Asp Met Pro Gly Thr Tyr Leu Asn Arg Ser AlaAsp Glu 465 470 475 480 Phe Asn Leu Ser 11 528 PRT Mus musculus 11 MetAla Trp Phe Gly Tyr Cys Leu Ala Leu Leu Ala Leu Thr Trp His 1 5 10 15Ser Ser Ala Tyr Gly Pro Asp Gln Arg Ala Gln Lys Lys Ile Ile Leu 20 25 30Gly Gly Pro Ile His Phe Gly Val Ser Ala Lys Asp Gln Asp Leu Lys 35 40 45Ser Arg Pro Glu Ser Val Glu Ile Arg Tyr Asn Phe Arg Phe Arg Trp 50 55 60Leu Gln Ile Phe Ala Ile Glu Glu Ser Ser Pro Ala Asn Met Thr Tyr 65 70 7580 Arg Ile Phe Thr Asn Thr Val Ser Lys Ala Leu Glu Ala Thr Leu Ser 85 9095 Phe Val Ala Gln Asn Lys Ile Asp Ser Leu Asn Leu Asp Glu Phe Cys 100105 110 Asn Cys Ser Glu His Ile Pro Ser Thr Ile Ala Val Val Ala Thr Gly115 120 125 Ser Gly Val Ser Thr Ala Val Ala Asn Leu Leu Gly Leu Tyr IleGln 130 135 140 Val Ser Ala Ser Ser Ser Arg Leu Ser Asn Lys Asn Gln PheLys Ser 145 150 155 160 Phe Leu Thr Ile Asn Asp Glu His Gln Ala Thr AlaMet Ala Asp Ile 165 170 175 Ile Glu Tyr Phe Arg Asn Trp Val Gly Thr IleAla Ala Asp Asp Asp 180 185 190 Arg Pro Gly Ile Glu Lys Phe Arg Glu GluAla Glu Glu Arg Asp Ile 195 200 205 Ile Asp Phe Ser Glu Leu Ile Ser GlnTyr Ser Asp Glu Glu Glu Ile 210 215 220 Gln Gln Val Val Glu Val Ile GlnAsn Ser Thr Ala Lys Ile Val Val 225 230 235 240 Ser Ser Gly Pro Asp LeuGlu Pro Leu Ile Lys Glu Ile Val Arg Arg 245 250 255 Asn Ile Thr Gly ArgIle Trp Leu Ala Glu Ala Ala Ser Ser Ser Leu 260 265 270 Ile Ala Met ProGlu Tyr Phe His Val Val Gly Thr Ile Gly Phe Gly 275 280 285 Leu Lys AlaGly Gln Ile Pro Gly Phe Arg Glu Phe Leu Gln Lys Val 290 295 300 His ProArg Lys Ser Val His Asn Gly Phe Ala Lys Glu Phe Trp Glu 305 310 315 320Glu Thr Phe Asn His Leu Gln Asp Gly Ala Lys Gly Pro Leu Pro Val 325 330335 Asp Thr Phe Val Arg Ser His Glu Glu Gly Gly Asn Arg Leu Leu Asn 340345 350 Ser Ser Thr Ala Phe Arg Pro Leu Thr Gly Asp Glu Asn Ile Asn Ser355 360 365 Val Glu Thr Pro Tyr Met Asp Tyr Glu His Leu Arg Ile Ser TyrAsn 370 375 380 Val Tyr Leu Val Ser Ile His Ala Gln Asp Ile Tyr Thr LeuPro Gly 385 390 395 400 Arg Gly Leu Phe Thr Asn Gly Ser Ala Asp Ile LysLys Val Glu Ala 405 410 415 Trp Gln Val Lys His Leu Arg His Leu Asn ThrAsn Asn Met Gly Glu 420 425 430 Gln Val Thr Glu Cys Asp Leu Val Gly AsnTyr Ser Ile Ile Asn Trp 435 440 445 His Leu Ser Pro Glu Asp Gly Ser IleVal Phe Lys Glu Val Gly Tyr 450 455 460 Tyr Asn Val Tyr Ala Lys Lys GlyGlu Arg Phe Ile Asn Glu Gly Lys 465 470 475 480 Ile Leu Trp Ser Gly PheSer Arg Glu Pro Phe Asn Ser Arg Asp Gln 485 490 495 Ala Thr Arg Lys GlyIle Ile Glu Gly Glu Pro Thr Phe Glu Ala Glu 500 505 510 Cys Pro Asp GlyGlu Tyr Ser Gly Glu Thr Asp Ala Ser Ala Asp Lys 515 520 525 12 500 PRTMus musculus 12 Phe Phe Pro Met Ile Phe Leu Glu Met Ser Ile Leu Pro ArgMet Pro 1 5 10 15 Asp Arg Lys Val Leu Leu Ala Gly Ala Ser Ser Gln ArgSer Val Ala 20 25 30 Arg Met Asp Val Ile Ile Gly Ala Ser Val His His GlnPro Pro Ala 35 40 45 Glu Lys Val Pro Glu Arg Lys Gly Glu Ile Arg Glu GlnTyr Ile Gln 50 55 60 Arg Val Glu Phe His Thr Leu Asp Lys Ala Asp Pro ValAsn Ile Thr 65 70 75 80 Ser Glu Ile Arg Ser Trp His Ser Ser Val Ala LeuGlu Gln Ser Ile 85 90 95 Glu Phe Ile Arg Asp Ser Leu Ile Ser Ile Arg AspGlu Lys Asp Gly 100 105 110 Leu Asn Arg Cys Leu Pro Asp Gly Gln Thr LeuPro Pro Gly Arg Thr 115 120 125 Lys Lys Pro Ile Ala Gly Val Ile Pro GlySer Ser Ser Val Ala Ile 130 135 140 Gln Val Gln Asn Leu Leu Gln Leu AspIle Gln Ile Ala Ser Ala Thr 145 150 155 160 Ser Ile Asp Ser Asp Lys ThrLeu Tyr Lys Tyr Phe Leu Val Val Ser 165 170 175 Asp Thr Leu Gln Ala ArgAla Met Leu Asp Ile Val Lys Arg Tyr Asn 180 185 190 Thr Tyr Val Ser AlaVal His Thr Glu Gly Asn Glu Ser Gly Met Asp 195 200 205 Ala Phe Lys GluLeu Ala Ala Gln Glu Gly Leu Ile Ala His Ser Asp 210 215 220 Lys Ile TyrSer Asn Ala Gly Glu Lys Ser Phe Asp Arg Leu Leu Arg 225 230 235 240 LysLeu Arg Glu Arg Leu Pro Lys Ala Arg Val Val Cys Cys Glu Gly 245 250 255Met Thr Val Arg Gly Leu Leu Ser Ala Met Arg Arg Leu Gly Val Val 260 265270 Gly Glu Phe Ser Leu Ile Gly Asp Gly Ala Asp Arg Asp Glu Val Ile 275280 285 Glu Gly Tyr Glu Val Glu Ala Asn Gly Ile Thr Ile Lys Leu Gln Ser290 295 300 Pro Glu Val Arg Ser Phe Asp Asp Tyr Phe Leu Lys Leu Arg LeuAsp 305 310 315 320 Thr Asn Thr Arg Asn Pro Trp Phe Pro Glu Phe Trp GlnHis Arg Phe 325 330 335 Gln Arg Leu Pro Gly His Leu Leu Glu Asn Pro AsnPhe Lys Lys Val 340 345 350 Thr Gly Asn Glu Ser Leu Glu Glu Asn Tyr ValGln Asp Ser Lys Met 355 360 365 Gly Phe Val Ile Asn Ile Ala Met His GlyGln Asn Met His His Ala 370 375 380 Leu Pro Gly His Val Gly Leu Asp AlaMet Lys Pro Ile Asp Gly Arg 385 390 395 400 Lys Leu Asp Phe Leu Ile LysSer Ser Val Gly Val Ser Gly Glu Glu 405 410 415 Val Trp Glu Lys Asp AlaPro Gly Arg Tyr Asp Ile Met Asn Leu Gln 420 425 430 Tyr Thr Glu Ala AsnArg Tyr Asp Tyr Val His Val Gly Thr Trp His 435 440 445 Glu Gly Val AsnIle Asp Asp Tyr Lys Ile Gln Met Asn Lys Ser Gly 450 455 460 Met Arg ValSer Glu Pro Leu Lys Gln Ile Lys Val Ile Arg Lys Gly 465 470 475 480 GluVal Ser Trp Ile Thr Ala Lys Glu Asn Glu Phe Val Gln Asp Glu 485 490 495Phe Thr Arg Ala 500

We claim:
 1. An isolated nucleic acid molecule comprising a nucleotidesequence that encodes the amino acid sequence shown in FIG. 1B.
 2. Theisolated nucleic acid molecule of claim 1 comprising the DNA sequence ofFIG. 1B.
 3. The isolated nucleic acid molecule of claim 2 comprising anucleotide sequence that encodes the amino acid sequence shown in FIG.1B.
 4. An isolated nucleic acid molecule comprising a nucleotidesequence that hybridizes to the nucleotide sequence of claim 1 or 2under stringent conditions and encodes a functionally equivalent geneproduct.
 5. An isolated nucleic acid molecule comprising a nucleotidesequence that hybridizes to the nucleic acid of claim 1 or 2 undermoderately stringent conditions and encodes a functionally equivalentT1R3 gene product.
 6. An isolated nucleic acid molecule that is a T1R3antisense molecule.
 7. An isolated polypeptide comprising the amino acidsequence of FIG. 1B.
 8. An isolated polypeptide comprising the aminoacid sequence encoded by a nucleotide sequence that hybridizes to thenucleotide sequence of claim 1 or 2 under stringent conditions andencodes a functionally equivalent gene product.
 9. An isolatedpolypeptide comprising the amino acid sequence encoded by a nucleotidesequence that hybridizes to the nucleotide sequence of claim 1 or 2under moderately stringent conditions and encodes a functionallyequivalent gene product.
 10. A purified fragment of a T1R3 proteincomprising a domain of the T1R3 protein selected from the groupconsisting of the amino terminal domain, transmembrane domain andcytoplasmic domain.
 11. A chimeric protein comprising a fragment of aT1R3 protein consisting of at least 6 amino acids fused via a covalentbond to an amino acid sequence of a second protein, in which the secondprotein is not a T1R3 protein.
 13. An antibody which is capable ofbinding a T1R3 protein. A recombinant cell containing the nucleic acidof claim 4 or
 5. 14. A method of producing a T1R3 protein comprisinggrowing a recombinant cell containing the nucleic acid of claim 4 or 5such that the encoded T1R3 protein is expressed by the cell, andrecovering the expressed T1R3 protein.
 15. A method for identifying acompound that induces the perception of a sweet taste comprising: (i)contacting a cell expressing the T1R3 channel protein with a testcompound and measuring the level of T1R3 activation; (ii) in a separateexperiment, contacting a cell expressing the T1R3 receptor protein witha vehicle control and measuring the level of T1R3 activation where theconditions are essentially the same as in part (i); and (iii) comparingthe level of activation of T1R3 measured in part (i) with the level ofactivation of T1R3 in part (ii), wherein an increased level of activatedT1R3 in the presence of the test compound indicates that the testcompound is a T1R3 inducer.
 16. A method for identifying a compound thatinhibits the perception of a sweet taste and/or promotes the perceptionof a sweet taste comprising: (i) contacting a cell expressing the T1R3receptor protein with a test compound in the presence of a sweet tastantand measuring the level of T1R3 activation; (ii) in a separateexperiment, contacting a cell expressing the T1R3 receptor protein witha sweet tastant and measuring the level of T1R3 activation, where theconditions are essentially the same as in part (i); and (iii) comparingthe level of activation of T1R3 measured in part (i) with the level ofactivation of T1R3 in part (ii), wherein a decrease level of activationof T1R3 in the presence of the test compound indicates that the testcompound is a T1R3 inhibitor.
 17. A method for identifying an inhibitorof sweet taste in vivo comprising: (i) offering a test animal the choiceof consuming either (a) a composition comprising a sweet tastant or (b)the composition comprising the sweet tastant as well as a testinhibitor; and (ii) comparing the amount of consumption of thecomposition according to (a) or (b), wherein greater consumption of thecomposition according to (a) has a positive correlation with an abilityof the test inhibitor to inhibit the perception of sweet tasteassociated with the tastant.
 18. A method for identifying an activatorof sweet taste in vivo comprising: (i) offering a test animal the choiceof consuming either (a) a control composition or (b) the compositioncomprising a test activator; and (ii) comparing the amount ofconsumption of the composition according to (a) or (b), wherein greaterconsumption of the composition according to (b) has a positivecorrelation with an ability of the test activator to activate theperception of sweet taste.
 19. A method of inhibiting a sweet tasteresulting from contacting a taste tissue of a subject with a sweettastant, comprising administering to the subject an effective amount ofa T1R3 inhibitor.
 20. A method of producing the perception of a sweettaste by a subject, comprising administering, to the subject, acomposition comprising a compound that acts as an activator of T1R3. 21.A method of producing the perception of a sweet taste by a subject,comprising administering, to the subject, a composition comprising acompound that acts as a sweetness activator.